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Carbohydrate utilization in selected strains of British Columbia chinook salmon Mazur, Carol Nelson 1990

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CARBOHYDRATE UTILIZATION BRITISH COLUMBIA IN SELECTED STRAINS OF CHINOOK SALMON by Carol Nelson Mazur B . S c . , M c G i l l U n i v e r s i t y , 1986 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES (Department of Animal Science) We accept th i s thes is as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA September 1990 © Caro l Nelson Mazur, 1990 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ANIMAL S C I E N C E The University of British Columbia Vancouver, Canada S e p t e m b e r 2 5 , 1 9 9 0 . Date DE-6 (2/88) ii Abstract D iges t ib l e carbohydrate i s commonly encountered by chinook salmon {Oncorhynchus tshawytscha) in p r a c t i c a l cu l ture d i e t s , although l i t t l e i s known regarding i t s u t i l i z a t i o n . This study was undertaken to examine (1) the e f fects of a high carbohydrate diet and (2) glucose tolerance in chinook salmon of selected B r i t i s h Columbia s t r a i n s . Year l ing chinook salmon of three s t ra ins were fed to s a t i a t i o n e i ther a high or a low carbohydrate d iet for 63 days. The d ie ts were isonitrogenous, and contained respect ive ly 30 % ge la t in i zed wheat starch or an equ ica lor i c amount of herr ing o i l . There was an o v e r a l l reduction in growth of chinook fed the high-carbohydrate d ie t over the 63-day feeding per iod . Although spec i f i c growth rates decl ined i n i t i a l l y in the high carbohydrate-fed groups, they were comparable to those of contro l groups in the f i n a l t h i r d of the t r i a l , ind ica t ing an adaptation response. Chinook fed the high carbohydrate d iet had increased carcass prote in and ash, and decreased carcass fat l eve ls r e l a t i v e to c o n t r o l s . Feed intake was general ly lower in these groups, and dif ferences in feeding response were observed between d ie ts and s t r a i n s . Although feed and energy e f f i c i e n c i e s were reduced in chinook fed the high carbohydrate d i e t , prote in u t i l i z a t i o n was comparable on the two d i e t s , ind ica t ing a prote in-spar ing effect of the carbohydrate. Consumption of the high carbohydrate diet led to s i g n i f i c a n t e levations in hepatosomatic indices (HSI) and iii l i v e r glycogen (LG) concentrat ions . In Quesnel chinook, LG leve l s exceeding 10 % d id not appear to have any detrimental e f fects on feeding, growth or hea l th . LG concentrations and HSI f e l l to basal l eve l s in a l l groups 21 days after feed withdrawal. Some s t r a i n di f ferences were evident . For example, Big Qualicum chinook fed the high carbohydrate diet exhibited the lowest l i v e r glycogen accumulation, highest rate of carcass fat depos i t ion , and best energy e f f i c i ency ra t ios r e l a t i v e to contro l groups, suggesting a di f ference in carbohydrate metabolism in th i s s t r a i n . On the other hand, Quesnel chinook exhibi ted the highest r e l a t i v e growth on the high carbohydrate d i e t . M o r t a l i t y , although unaffected by d ie t in the Quesnel and Robertson Creek chinook, appeared to be higher in high carbohydrate-fed Big Qualicum chinook. In the second part of the study, chinook salmon subjected to an o r a l glucose tolerance test displayed pronounced and pers is tent hyperglycaemia, ind ica t ive of poor glucose to lerance . S tra in di f ferences were evident in the magnitude of response. Accl imation to a high carbohydrate d iet p r i o r to tes t ing resul ted in a s i g n i f i c a n t l y reduced e levat ion of blood glucose, i n d i c a t i n g an adaptation response. While plasma glucose concentrations approached 500 mg/dl in some t r i a l s , plasma i n s u l i n concentrations exhibi ted a two-fold r i s e , with i n d i s t i n c t peaks. Plasma glucose and plasma i n s u l i n concentrations were poorly c o r r e l a t e d , ind icat ing that glucose i s a poor i n s u l i n secretagogue in chinook salmon. iv TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i i LIST OF FIGURES ix ACKNOWLEDGEMENTS x 1 INTRODUCTION 1 2 LITERATURE REVIEW 4 2.1 Carbohydrate i n Salmonid D i e t s : L i t e r a t u r e Recommendations and Controversy 4 2.2 E n e r g e t i c s 6 2.3 D i g e s t i b i l i t y 7 2.4 Carbohydrate Energy Values 9 2.5 Temperature E f f e c t s 10 2.6 F i s h S i z e 1 1 2.7 Carbohydrate Source 12 2.8 I n d i g e s t i b l e Carbohydrate ( E f f e c t s of F i b e r ) 14 2.9 Species Comparisons ( F i s h ) 16 2.9.1 S t r a i n Comparisons . 17 2.10 Carbohydrate S t r u c t u r e 19 2.11 Carbohydrate D i g e s t i o n and Absorption 19 2.12 Carbohydrate Metabolism 20 2.12.1 G l y c o l y s i s and Gluconeogenesis 21 2.12.1.1 Enzyme Adaptation 25 2.12.2 Glycogen S y n t h e s i s and Breakdown 26 2.12.2.1 L i v e r glycogen S t u d i e s 30 2.13 Endocrine C o n t r o l 31 2.13.1 I n s u l i n 31 2.13.2 Glucagon 35 2.13.3 T h y r o i d Hormone 35 2.14 O r a l Glucose T o l e r a n c e 35 3 GENERAL MATERIALS AND METHODS 39 3.1 H i s t o r y and Maintenance of Experimental F i s h 39 4 EXPERIMENT 1. The E f f e c t s of Feeding a High 42 Versus Low Carbohydrate D i e t on Growth, Body Composition, Feed and P r o t e i n U t i l i z a t i o n , L i v e r Glycogen C o n c e n t r a t i o n and L i v e r Weight i n S e l e c t e d S t r a i n s of B. C. Chinook Salmon. V 4.1 MATERIALS AND METHODS 42 4. 1 . 2 Diet Preparation and Composition 42 4. 1 . 3 F i s h Select ion and Tank A l l o c a t i o n 45 4. 1 . 4 Start of Experiment 46 4. 1 . 5 Proximate Body Composition 47 4. 1 . 6 L i v e r Glycogen Determination 49 4. 1 . 7 S t a t i s t i c a l Analyses 50 4. 2 RESULTS 51 4. 2. 1 Proximate Analyses of Diets and Prote in 51 Sources 4. 2. 2 Growth and Condit ion Factor 51 4. 2. 3 Carcass Composition 60 4. 2. 3 . 1 Growth in Terms of Protein and Fat 64 4. 2. 4 Feed Intake and E f f i c i e n c y Indices 64 4. 2. 5 Prote in U t i l i z a t i o n 70 4. 2. 6 L i v e r Glycogen and Hepatosomatic Index 73 4. 2. 7 M o r t a l i t y 76 4. 3 DISCUSSION 78 4. 3. 1 General 78 4. 3. 2 Growth Rates 79 4. 3. 3 Carcass Composition 80 4. 3. 3 . 1 Growth in Terms of Carcass Composition 80 4. 3. 4 Feed Intake and Feeding Response 81 4. 3. 5 Feed and Energy E f f i c i e n c y 82 4. 3. 6 U t i l i z a t i o n of Prote in 85 4. 3. 7 L i v e r Effects 86 4. 3. 8 Morta l i t y 87 4. 4 CONCLUSIONS (Experiment 1) 89 5 EXPERIMENT 2 92 5. 1 Part i ) Oral Glucose Tolerance in Selected Strains of B. C. Chinook Salmon. 92 5. 1 . 1 MATERIALS AND METHODS 92 5. 1 . 1 .2 Tank Preparation and Fish A l l o c a t i o n 92 5. 1 . 1 .3 Glucose Adminis trat ion: Prel iminary T r i a l 93 5. 1 . 1 .4 Glucose Tolerance T r i a l s 96 5. 1 . 1 .5 Experimental Sampling 97 5. 1 . 1 .6 Blood Sampling Technique 97 5. 1 . 1 .7 Plasma Glucose Determination 98 5. 1 . 1 .8 Plasma Insu l in Radioimmunoassay 99 5. 1 . 1 .9 S t a t i s t i c a l Analys i s 100 vi 5.2 Part i i ) Oral Glucose Tolerance in Chinook Salmon Acclimated to High and Low Carbohydrate Diets Respectively 101 5.2.1 MATERIALS AND METHODS 101 5.2.1.1 Glucose Tolerance T r i a l s 101 5.2.1.2 S t a t i s t i c a l Analys i s 102 5.3 RESULTS 103 5.3.1 Part i ) Oral Glucose Tolerance in Chinook Salmon of Selected Stra ins 103 5.3.1.1 Plasma Glucose Response 103 5.3.1.2 Plasma Insul in Response 106 5.3.2 Part i i ) Ef fect of Pre-Test Diet on Oral Glucose Tolerance 107 5.3.2.1 Plasma Glucose Response 110 5.3.2.2 Plasma Insul in response 110 5.4 DISCUSSION 112 5.4.1 Oral Glucose Tolerance: General 112 5.4.1.1 Part i ) Oral Glucose Tolerance in Chinook Salmon of Selected Stra ins 114 5.4.2.1 Part i i ) Ef fect of Pre-Test Diet on Oral Glucose Tolerance 117 5.5 CONCLUSIONS (Experiment 2) 119 6 REFERENCES 121 vii LIST OF TABLES Table 1 - Composition of test d ie t s as fed. 43 Table 2 - Calculated metabolizable energy values of test d iets on a moisture-free bas i s . 44 Table 3 - Proximate analyses of d i e t s . 52 Table 4 - Proximate analyses of prote in sources for experimental d ie t s (dry matter b a s i s ) . 53 Table 5 - Mean body weights of treatment groups at 21-day in terva l s during the experiment. 55 Table 6 - Average body weight gains (BWG) and percent BWG of treatment groups at the end of the 63-day feeding per iod . 57 Table 7 - Spec i f i c growth rates of treatment groups. 58 Table 8 - Mean condit ion factors in treatment groups, i n i t i a l l y and at the end of the 63-day feeding per iod . 61 Table 9 - Proximate analyses of whole f i s h . 62 Table 10 - Instantaneous prote in gain (IPG) and instantaneous l i p i d gain (ILG) in treatment groups over the 63-day feeding p e r i o d . 65 Table 11 - Mean prote in weights of chinook in treatment groups, i n i t i a l l y and at the end of the 63-day feeding t r i a l . 66 Table 12 - Feed intake (FI ) , feed e f f i c i ency and energy e f f i c i ency (EE) ra t ios in the treatment groups at the end of the 63-day feeding per iod . Table 13 - Protein e f f i c i ency ra t ios (PER) and productive prote in values (PPV) in treatment groups over the 63-day feeding p e r i o d . Table 14 - Mean percent l i v e r glycogen concentrations and hepatosomatic indices (HSI) in treatment groups ( i n i t i a l l y , during feeding and 21 days post-feed withdrawal. 67 71 74 Table 15 - Tota l m o r t a l i t i e s in treatment groups over the 63-day feeding per iod . 77 viii Table 16 - Mean plasma glucose and i n s u l i n concen-tra t ions in 3 s tra ins of chinook salmon subjected to an o r a l glucose tolerance test (GTT). 104 Table 17 - Mean plasma glucose and i n s u l i n concen-tra t ions in Capilano chinook salmon subjected to an o r a l glucose tolerance test (GTT) after feeding low and high carbohydrate pre- tes t d ie ts respec t ive ly . 108 ix LIST OF FIGURES Figure 1 - Glucose Metabolism :g lyco ly s i s and gluconeogenesis pathways. 22 Figure 2 - L iver Glycogen Synthesis and Breakdown. 27 Figure 3 - Growth of Chinook Salmon of Three Strains Fed Low VS High Carbohydrate Diets over a 63-Day Per iod . 54 Figure 4 - Twenty-One Day Interval Spec i f i c Growth Rates of Chinook Salmon of Three Stra ins Fed Low VS High Carbohydrate D i e t s . 59 Figure 5 - Ef fec t of Dietary Carbohydrate on Body Composition of Chinook Salmon of Three Stra ins Fed Low or High Carbohydrate Diets for 63 Days. 63 Figure 6 - Prote in Weights of Chinook Salmon Fed Low or High Carbohydrate Diets for 63 Days. 68 Figure 7 - Feed E f f i c i e n c y and Energy E f f i c i e n c y Ratios of Chinook Salmon of Three Stra ins Fed Low or High Carbohydrate Diets for 63 Days. 69 Figure 8 - Prote in E f f i c i e n c y Ratios and Productive Protein Values of Chinook Salmon of Three Stra ins Fed Low or High Carbohydrate Diets for 63 Days. 72 Figure 9 - Ef fect of Dietary Carbohydrate on L iver Glycogen Level in Chinook Salmon of Three S t r a i n s : During Feeding and Subsequent to Feed Withdrawal. 75 Figure 10 - Prel iminary Comparison T r i a l of Glucose Adminis trat ion: Capsules VS So lu t ion . 95 Figure 11 - Oral Glucose Tolerance Test Response in Chinook Salmon of Selected B. C. S t r a i n s . 105 Figure 12 - Ef fect of Pre-Test Diet on Oral Glucose Tolerance in Capilano Chinook Salmon. 109 X ACKNOWLEDGEMENTS This research was supported through a Department of F i s h e r i e s and Oceans grant to Dr . D. A. Higgs. I would l i k e to thank Dr. B. E . March and Dr. D. A. Higgs for the continued support, guidance and commitment they have shown throughout th i s pro jec t . I would also l i k e to thank Bakhshish Dosanjh for his valued guidance in laboratory analyses. The generous assistance of Dr . E . P l i se t skaya in quanti fy ing the plasma i n s u l i n values for th i s research is great ly appreciated. I would l i k e to thank Brian Toy for his invaluable assistance in many aspects of th i s study. I would a lso l i k e to thank members of the facul ty and s taf f and students at U . B . C . for the ir ass is tance , and for making my studies most enjoyable. F i n a l l y , I would l i k e to thank my husband C a r l for his love, encouragement and sense of humour throughout the p r o j e c t . 1 1 INTRODUCTION Salmonids are among the 85 % of known f i s h s p e c i e s that are c a r n i v o r e s , and t h e i r n a t u r a l d i e t c o n t a i n s very l i t t l e c arbohydrate. The p r o t e i n requirement of f i s h , when expressed as a percentage of the d i e t , i s 2 to 4 times that of t e r r e s t r i a l homeotherms and d i e t a r y p r o t e i n energy to t o t a l energy r a t i o i s approximately 1 : 2 ( T y t l e r and Calow, 1985). Looking at t h i s from another p e r s p e c t i v e , f i s h r e q u i r e l e s s energy than warm-blooded animals. T h i s i s because the p o i k i l o t h e r m i c f i s h does not have to maintain a body temperature d i f f e r e n t from that of the environment, r e q u i r e s l e s s energy f o r locomotion (Cowey, 1980) and can e f f e c t i v e l y use p r o t e i n both as a source of amino a c i d s f o r s y n t h e s i s of body t i s s u e p r o t e i n s and as an energy source. In f a c t , f i s h use p r o t e i n energy p r e f e r e n t i a l l y over carbohydrate energy f o r metabolism (Smith e t - a l . , 1978; Cowey and Luquet, 1983). Most of t h e i r waste n i t r o g e n i s e x c r e t e d as ammonia v i a the g i l l s ; an e n e r g e t i c a l l y inexpensive process i n comparison to urea or u r i c a c i d p r o d u c t i o n . T h i s allows the f i s h to r e t a i n more energy per u n i t p r o t e i n i n g e s t e d than other animals. Salmonids e x h i b i t a number of l i m i t a t i o n s i n t h e i r a b i l i t y to u t i l i z e d i e t a r y carbohydrate. They are g e n e r a l l y c o n s i d e r e d to be d i a b e t i c , as i n d i c a t e d by t h e i r i n a b i l i t y to c o n t r o l blood glucose and poor i n s u l i n s t a t u s (Palmer and Ryman, 1972). Moreover, they possess low a c t i v i t y l e v e l s of key enzymes i n v o l v e d i n carbohydrate metabolism, and d i s p l a y a l i m i t e d a b i l i t y f o r enzymatic a d a p t a t i o n ( H i l t o n and S l i n g e r , 2 1982). Consumption of high d ie tary leve ls of d i g e s t i b l e carbohydrate leads to excessive l i v e r glycogen accumulation (Hi l ton and Atkinson, 1982; Bergot, 1979a; Palmer and Ryman, 1972; P h i l l i p s et al., 1948). In formulating p r a c t i c a l d ie t s for cul tured salmonids, carbohydrate i s important for several reasons. For instance, on a cost per unit weight bas i s , i t i s the least expensive source of d ie tary energy for use in sparing expensive d ie tary prote in for growth. A l s o , carbohydrate in the form of starch is a major const i tuent of inexpensive plant prote in sources which are employed to p a r t i a l l y replace expensive marine f i s h prote in sources. Diet processing methods which involve heat and pressure p a r t i a l l y hydrolyze the starch and increase i t s b i o l o g i c a l a v a i l a b i l i t y . Carbohydrate i s often used as a f i l l e r in d ie t s and also as a binder to give good p e l l e t s t a b i l i t y and i n t e g r i t y . The l i t e r a t u r e contains considerable controversy regarding the optimal d ie tary carbohydrate l e v e l for salmonids and other f i s h species . Research recommendations range from 9% to 30% as the maximum acceptable d ietary l e v e l of d i g e s t i b l e carbohydrate for salmonids (Hil ton et al. 1987a; Beamish et at., 1986; H i l t o n et al., 1982; Bergot, 1979a; Buhler and Halver , 1961; P h i l l i p s et al . , 1948). The determination of an optimal l e v e l of d ie tary carbohydrate i s hindered by the fact that so many var iables a f fec t i t s d iges t ion , metabolism and u t i l i z a t i o n by the f i s h . These include: carbohydrate source and form, d ie tary l e v e l , 3 processing of the diet ( i e . steam versus extrusion p e l l e t i n g ) , water temperature and s a l i n i t y , season, f i s h species and subspecies, age, l i f e stage and s ize of f i s h , feeding l e v e l and duration on a p a r t i c u l a r d i e t , s tress , and other environmental and d ie tary fac tors . The n u t r i t i o n a l needs of post juveni le chinook in sea water are v i r t u a l l y unknown even though th i s species i s presently preferred for cu l ture in B r i t i s h Columbia. Diet formulations current ly in use have been derived p r i m a r i l y from research on rainbow trout (Oneorhynchits mykiss) and A t l a n t i c salmon (Salmo salar). In a d d i t i o n , a wide range of carbohydrates has been tested in salmonid d i e t s : from simple sugars such as glucose, which i s r ead i ly absorbed, to complex carbohydrates, e spec ia l l y starches, which are highly var iab le in the i r d igest ion and absorpt ion. Carbohydrate u t i l i z a t i o n in chinook salmon has been examined in only one study (Buhler and Halver , 1961). The l a t t e r authors reported that chinook exhib i t a marked a b i l i t y to u t i l i z e d ie tary carbohydrate, and superior u t i l i z a t i o n of lower molecular weight carbohydrates. This study was undertaken in order to examine: (1) the e f fects of high versus low carbohydrate d iets on growth, feed and prote in u t i l i z a t i o n , and l i v e r glycogen in chinook of se lected B . C . stocks held in sea water, and (2) o r a l glucose tolerance as an index of carbohydrate u t i l i z a t i o n in sea water acclimated chinook of (a) selected B . C . stocks and (b) selected stocks pre-accl imated to high and low carbohydrate d i e t s , re spec t ive ly . 4 2 LITERATURE REVIEW 2.1 Carbohydrate in Salmonid Die t s : L i t e r a t u r e Recommendations and Controversy There i s considerable debate in the l i t e r a t u r e regarding the optimum dietary l e v e l of d iges t ib l e carbohydrate for salmonids. Numerous studies have been conducted on d i f f erent f i s h species examining growth, feed e f f i c i e n c y , d i g e s t i b i l i t y , enzyme adaptat ion, glucose to lerance , metabolism and other parameters in order to assess carbohydrate u t i l i z a t i o n . H i l t o n et al. (1982) made a d i s t i n c t i o n between maximum to lerable d i g e s t i b l e carbohydrate and optimum leve l of d i g e s t i b l e carbohydrate. The former term refers to the "level which can be e f f e c t i v e l y digested and metabolized . . .w i thout causing growth impairment or increase in morta l i ty rate" and the l a t t e r "the l eve l that cannot only be e f f i c i e n t l y digested and metabolized but a lso e f f i c i e n t l y u t i l i z e d as an energy source". H i l t o n et al. (1987a) concluded that salmonids cannot e f f e c t i v e l y u t i l i z e more than 14% (140 g/kg) d i g e s t i b l e carbohydrate in the d i e t , based on feeding t r i a l s with rainbow t r o u t . Above th i s l eve l no further adaptation of key carbohydrate ca tabo l i z ing enzymes occurred (Hil ton and Atkinson, 1982). In 1987, Hi l ton demonstrated s i g n i f i c a n t growth depression in rainbow trout fed a 25% glucose diet with 43% prote in as compared to an isonitrogenous isoenergetic contro l d ie t (Hil ton et al . , 1987b). P h i l l i p s 5 et al. (1948) found that young brook trout (Salvelinus fontinalis), supplied with more than 9% d i g e s t i b l e carbohydrate in the ir d i e t , exhibi ted growth reduct ion, high-glycogen l i v e r s and increased m o r t a l i t y . Reports by other invest igators have been contrad ic tory , ind ica t ing that 20% (200 g/kg) or more of d i g e s t i b l e carbohydrate i s e f f e c t i v e l y u t i l i z e d . For example, Buhler and Halver (1961) reported that up to 48% of dextr in or 20% of low molecular weight sugars could be to lerated by chinook salmon (Oncorhynchus tshawytscha) with no patho log ica l consequences or growth depression. Moreover, Luquet (1971) noted that rainbow trout fed a 30% prote in 50% raw corn starch d ie t exhibi ted the same growth as, and better prote in u t i l i z a t i o n than those fed d ie ts containing twice as much p r o t e i n . Further , Bergot (1979a) reported e f f ec t ive u t i l i z a t i o n of a 30% glucose diet by rainbow trout when d ie tary prote in content was 45%. However, when the prote in l e v e l was lowered to 30%, growth depression resu l t ed . S i m i l a r l y , Luquet et al. (1975) found a dec l ine in sucrose u t i l i z a t i o n when prote in l eve l was decreased from 55% to 35%. In a 1986 study by Beamish et al., rainbow trout fed a 30% glucose diet had s i g n i f i c a n t l y lower body weights and carcass f a t , but s i g n i f i c a n t l y higher feed conversion and carcass prote in than trout fed an isonitrogenous diet with 18% l i p i d . Based on evidence in the l i t e r a t u r e , Cowey & Sargent (1979) stated that carbohydrate (dextrin or starch) l eve ls 6 of approximately 25% of the d ie t are as e f f ec t ive as equ ica lor i c amounts of fat for energy for a v a r i e t y of f i s h species inc luding channel c a t f i s h , rainbow trout and p la i ce (NRC, 1981). H i l t o n , however, recommended that the maximum "tolerable" l eve l of d i g e s t i b l e carbohydrate for salmonids is approximately 200 g/kg d i e t , while the "optimal" l e v e l i s in the neighborhood of 140 g/kg (Hi l ton and Atkinson, 1982). Disagreement in the l i t e r a t u r e i s not s u r p r i s i n g , as many factors complicate the determination of an idea l carbohydrate l e v e l . Species di f ferences make i t d i f f i c u l t to extrapolate experimental re su l t s from one species to another. A l s o , the compositions of the experimental d i e t s , d i g e s t i b i l i t i e s of the test carbohydrate sources, and water temperatures have varied widely between s tudies . 2.2 Energetics i t i s e s sent ia l to understand the energy requirements of salmonids to minimize the use of d ie tary prote in for energy by these species . The prote in - spar ing act ion of l i p i d s has been confirmed in several f i sh species (Cho and Kaushik, 1985; Beamish and Medland, 1986), but the energy value of d i g e s t i b l e carbohydrate i s s t i l l in question (Kaushik et al., 1989). Many studies indicate that f i s h have a l i m i t e d a b i l i t y to adapt to increased d ie tary carbohydrate (Cowey et al . , 1977a; Cowey et al., 1977b; Furu ich i and Yone, 1980) and that increasing carbohydrate can spare prote in (Pieper and P f e f f e r , 1979; Shimeno et al., 7 1979, 1985). F i s h are said to eat to sa t i s fy the ir energy needs (Kaushik and Luquet, 1984). Therefore the d ietary protein:energy (P/E) r a t i o i s c r i t i c a l for maximizing the use of prote in for growth. The u t i l i z a t i o n of glucose for energy depends, in large part , on the l eve l s of a l t ernat ive energy sources in the diet (Hi l ton et al., 1982) and the d ie tary prote in content (Bergot, 1979a). H i l t o n et al. (1982) suggested that while salmonids may ' t o l e r a t e ' glucose l eve l s in excess of 25%, the sugar is probably not being e f f i c i e n t l y used for energy. 2.3 D i g e s t i b i l i t y Carbohydrate d i g e s t i b i l i t y can be a major obstacle in determining the appropriate d ie tary carbohydrate l e v e l for salmonids. The d i g e s t i b i l i t y of carbohydrate depends on many fac tors , inc luding carbohydrate s tructure , source, l e v e l of incorporat ion in the d i e t , processing technique, f i s h species and method of feca l c o l l e c t i o n (Hi l ton and S l i n g e r , 1983; Spannhof and Plantikow, 1983; Takeuchi el al. 1979; Bergot and Breque, 1983). The degree of polymerization of a carbohydrate i s the main element a f f ec t ing i t s d i g e s t i b i l i t y . The monosaccharide glucose is completely absorbed. By contrast , whereas complex highly branching carbohydrates are not well digested by most f i s h , inc luding rainbow trout (Singh and Nose, 1967; Bergot, 1979a), which are poor starch digesters (Spannhof and Plantikow, 1983). Studies indicate that the 8 d i g e s t i b i l i t y of raw starch i s below 50% ( P h i l l i p s et al., 1967; Bergot and Breque, 1983). Carbohydrate d i g e s t i b i l i t y a lso decreases with increasing dietary l e v e l , and the amount of starch in a d iet can af fect the absorption of other carbohydrates inc luding simple sugars. While the d i g e s t i b i l i t y of glucose i s r e l a t i v e l y unaffected by i t s l e v e l in the d ie t (Hi l ton et al . , 1982), the d i g e s t i b i l i t i e s of more complex carbohydrates such as starch and dextr in are negatively corre la ted with d ie tary l e v e l (Singh and Nose, 1967). Inaba et al. (1963) found the d i g e s t i b i l i t y of cooked wheat a-starch to be 90% and 48.2% at d ie tary l eve l s of 11.5% and 40%, respec t ive ly , in rainbow trout (from Singh and Nose, 1967). When raw starch is exposed to heat and pressure during processing i t becomes g e l a t i n i z e d , or p a r t i a l l y hydrolyzed. G e l a t i n i z a t i o n of starch great ly improves i t s d i g e s t i b i l i t y (Bergot and Breque, 1983) making i t s e f f i c i e n c y as an energy source for trout comparable to l i p i d s or prote in (Pieper and Pfe f f er , 1980a, 1980b). This increased a v a i l a b i l i t y i s r e f l e c t ed by elevated blood glucose l e v e l s , increased hepatosomatic indices (HSI), improved feed conversion and prote in e f f i c i ency ra t ios (Kaushik and de O l i v a Te les , 1985). Bergot and Breque (1983) reported a 90% d i g e s t i b i l i t y of g e l a t i n i z e d corn starch at a d ie tary l e v e l of 30% in rainbow trout . Other invest igat ions on rainbow trout indicate marked improvements in d i g e s t i b i l i t y and 9 energy a v a i l a b i l i t y of starch by extrusion processing or cooking (Kaushik and O l i v a Te les , 1985; Inaba et al., 1963). Carbohydrate d i g e s t i b i l i t y var ies great ly among d i f f erent species of f i s h . It has been demonstrated in carp that the d i g e s t i b i l i t i e s of dextr in (Takeuchi et al . 1979) and starch (Shimeno et al., 1979; Chiou and Ogino, 1975) at d ie tary l eve l s ranging from 10 to 40% are f a i r l y stable and much higher than those found in carvivorous species . In contrast , d i g e s t i b i l i t y of starch by carnivores such as the y e l l o w t a i l (Shimeno et al . , 1979) and rainbow trout (Singh and Nose, 1967; Inaba et al., 1963) s i g n i f i c a n t l y dec l ines with increasing d ie tary l e v e l , and great ly reduces the d i g e s t i b i l i t y of d ie tary p r o t e i n . 2.4 Carbohydrate Energy Values Caution must be exercised in ass igning energy values to carbohydrates in f i sh d ie ts (Job l ing , 1983). H i l t o n et al . (1987a) recommended that the use of d i g e s t i b l e energy (DE) and metabolizable energy (ME) values , which tend to overestimate productive energy values , be discontinued for determining u t i l i z a b l e energy of trout d i e t s . They proposed the net energy (NE) value as an a l t e r n a t i v e , which i s the percentage of gross energy of a carbohydrate that i s retained in the carcass . These invest igators ca l cu la ted NE values for glucose and starch in rainbow trout , each fed at a d ie tary l e v e l of 25%, to be approximately 25% of the glucose gross energy and 13% of the starch gross energy. 10 These values were considerably lower than expected, e spec ia l ly for glucose which i s almost completely absorbed. Although NE values may be more tenable, they are not f ixed and can be a l t ered by many factors that af fect carbohydrate d i g e s t i b i l i t y . One cannot consider only the ca lcu la ted energy values of carbohydrates. Carbohydrates have been shown to have an effect on voluntary feed intake. Studies show that raw s tarch , with i t s low d i g e s t i b i l i t y , acts as bulk in the d i e t , increas ing food intake (Hi l ton and S l i n g e r . , 1983; Kaushik and de O l i v a Te les , 1985). On the other hand, f i s h fed d ie t s containing glucose exhibi t a reduced feed intake which contr ibutes to t h e i r growth depression (Hi l ton and S l i n g e r , 1987a). Poor glucose tolerance and prolonged hyperglycaemia may af fect appet i te . 2.5 Temperature Ef fec ts As environmental temperature r i s e s or f a l l s , the f i s h ' s metabolism respect ive ly increases or decreases, and u t i l i z a t i o n of energy changes. Metabolic rate i s not d i r e c t l y re la ted to temperature, however, as adaptation occurs, lessening the d i f ference in responses between temperature extremes (Hochachka and Hayes, 1961). Routes of energy metabolism have also been shown to change with temperature. Hochachka and Hayes (1961), using 1 ^ C l a b e l l e d glucose, demonstrated that during co ld accl imation in brook t rout , the pentose phosphate pathway is 11 act ivated and fat synthesis i s enhanced. At higher temperatures, more glucose passes through the Embden-Meyerhof (or g lyco ly s i s ) pathway. S i m i l a r l y , H i l t o n et al. (1982) found a higher a c t i v i t y of glucose 6-phosphate dehydrogenase (G6PDH) (the enzyme leading into the pentose phosphate pathway) in 11°C acclimated rainbow trout versus those held at 15°C. L iver glycogen accumulation and l i v e r weight : body weight r a t i o (LBW) increase as temperature decreases (Hi l ton et al., 1982; H i l t o n , 1982; P h i l l i p s et al., 1966), and a longer fas t ing period i s required for l i v e r glycogen to f a l l at lower temperatures ( H i l t o n , 1982). Seibert (1985) concluded that gluconeogenesis i s the predominant source of glucose at lower temperatures, whereas glycogenolysis i s enhanced at higher temperatures. Although i t has been shown that the absorption of glucose i s not s i g n i f i c a n t l y a l t ered by temperature (Hi l ton et al. 1982), starch digest ion i s slower at lower temperatures, reducing the appearance rate (and hence uptake) of l ibera ted glucose. 2.6 F i s h Size The basic r e la t i onsh ip between body weight and metabolic rate i s described by the equation Y = a w ^ . 8 ) f where Y is the metabolic rate , W is the body weight and a i s a constant which var ies with f i s h species and temperature (Halver, 1989). Thus as weight increases, energy 12 requirement for metabolism decreases in r e l a t i o n to body weight. Tolerance to and u t i l i z a t i o n of carbohydrate are reported to increase, and prote in requirement decrease, with f i s h s ize (Rychly, 1980; Austreng et al . , 1977; P h i l l i p s , et al . , 1948). 2.7 Carbohydrate Source The type or source of carbohydrate present in a f i s h diet has a major bearing on how access ible i t s energy i s to the f i s h . In add i t i on , the p r o f i l e s and a c t i v i t y l eve l s of carbohydrate d igest ive enzymes vary among f i s h species . There must be a balance between the rate at which carbohydrate becomes ava i lab l e for metabolism ( i e . enters the c i r c u l a t i o n ) and i t s rate of u t i l i z a t i o n . It has generally been reported that f i s h grow better on higher molecular weight carbohydrates ( i e . starches) than on lower molecular weight sugars. It has been hypothesized that when high molecular weight carbohydrates are ingested, there i s a delay in absorption of glucose, which increases i t s u t i l i z a t i o n by al lowing more time for the a c t i v a t i o n of carbohydrate metabolism enzymes and i n s u l i n l eve l s to r i s e (Pieper and Pfe f f er , 1980b; Furu ich i and Yone, 1982a). There are numerous invest igat ions which lend support to th i s hypothesis . Decreased e f f i c i e n c y of u t i l i z a t i o n of simple sugars r e l a t i v e to g e l a t i n i z e d corn s tarch , p a r t i c u l a r l y at higher d ie tary l e v e l s , has been demonstrated in rainbow 13 trout (Pieper and Pfe f f er , 1979, 1980b). In a 1982 study by Akiyama, Murai and Nose, chum salmon fry (Oncorhynchus keta) fed g e l a t i n i z e d potato starch at a d ie tary l e v e l of 20% exhibi ted better feed and prote in e f f i c i e n c i e s than those fed several lower molecular weight carbohydrates, including glucose and d e x t r i n . Y e l l o w t a i l fed p u r i f i e d d ie t s containing 20% glucose exhibited hyperglycaemia, g lycosur ia , and s i g n i f i c a n t reductions in growth and feed e f f i c i e n c y . Starch, on the other hand, was absorbed more gradually al lowing i t to be p a r t i a l l y u t i l i z e d ( F u r u i c h i , T a i r a and Yone, 1986). A study on channel c a t f i s h indicated that low molecular weight sugars were not u t i l i z e d for energy, and indeed t h e i r d ie tary inc lus ion l e v e l led to decreased growth and prote in depos i t ion , while dextr in and corn starch served as good energy sources (Wilson and Poe, 1987). Furuich i and Yone (1982b) tested the r e l a t i v e u t i l i z a t i o n of glucose, dextr in and a-starch in carp and red sea bream. Growth and feed e f f i c i e n c y in carp were best with the starch d i e t , followed by d e x t r i n , then glucose. Red sea bream exhibited no s i g n i f i c a n t growth di f ferences between the three carbohydrate sources used and feed e f f i c i ency was only s l i g h t l y higher in the s tarch-fed f i s h . Several reports appear to be contradictory to those c i t e d above. For instance, Buhler and Halver (1961) found that young chinook salmon fed d ie ts containing 20% of d i f f eren t carbohydrates grew better on the lower molecular weight, r e a d i l y absorbed sugars. Hung et al. (1989) 14 reported s imi lar resu l t s in juveni le white sturgeon (Aci pens er transmontanus) fed p u r i f i e d d ie t s containing 27.2% of various carbohydrates. F i sh fed maltose and glucose d ie t s exhibi ted superior growth to those fed d ie t s containing dextr in or s tarch . The cont inual feeding strategy used in th i s study may have a l l e v i a t e d plasma glucose accumulation, as suggested by the lack of hyperglycaemia in the glucose- and maltose-fed f i s h . In Bergot's 1979 inves t iga t ion , rainbow trout fed a diet containing 30% of glucose exhibi ted superior growth, feed conversion and protein e f f i c i e n c y r a t i o to those fed a natural s tarch d i e t . .2.8 Indiges t ib le Carbohydrate ( The Ef fec ts of Fiber) Commercial and experimental f i sh d iets often contain a s i g n i f i c a n t amount of f i b e r . This "fiber" could be any number of ind iges t ib l e or complex carbohydrates inc luding c e l l u l o s e , hemicel lulose , l i g n i n , pec t in , and raw starch (Bromley and Adkins, 1984). In experimental f i s h d ie ts f iber i s used as a f i l l e r , to make up the bulk of d ie ts when manipulating leve ls of other nutr i ent s , or as a binder. In commercial or p r a c t i c a l formulations much of the f iber or ig inates from the inc lus ion of plant prote in sources. Studies show that f iber can affect the uptake and u t i l i z a t i o n of many d ie tary nutr ients inc luding d i g e s t i b l e carbohydrate. For example, f iber has been shown to reduce the in vitro i n t e s t i n a l absorption of glucose and the 15 u t i l i z a t i o n of dietary dextrin in T i l a p i a (Oreochromis sp.) (Shiau et al . , 1989). The physiological effects of fiber remain poorly understood in f i s h . Fiber also a l t e r s the rate of gastric evacuation. While fiber sources such as carboxymethylcellulose or ce l l u l o s e have been shown to increase the speed of stomach emptying (Shiau et al ., 1989; Jobling, 1981; Hilton et al., 1983) , other "viscous" fibers such as guar gum and pectin slow down evacuation (Shiau et al., 1989). Hilton, Atkinson and Slinger (1983) demonstrated s i g n i f i c a n t growth depression in juvenile rainbow trout fed diets containing 10 or 20% a-cellulose. Although the trout exhibited some adaptation to the f i b e r , by hypertrophy of the stomach and increased feed intake, i t was not s u f f i c i e n t to compensate for the increased bulk. Bromley and Adkins (1984), on the other hand, found that juvenile rainbow trout can compensate for up to 30% dietary c e l l u l o s e by increasing their feed intake. Growth and nutrient and protein conversion e f f i c i e n c i e s were not affected adversely. The trout stomachs became heavier only at 40% to 50% dietary c e l l u l o s e l e v e l s . These investigators suggested that i f increased bulk of the stomach i s an adaptation to f i b e r , then perhaps smaller f i s h are more limited in their a b i l i t y to handle f i b e r . Chinook salmon fed 50% ce l l u l o s e diets exhibited reduced feed intake and lowered protein e f f i c i e n c y (Buhler and Halver, 1961). Channel c a t f i s h also reduced the i r feed intake at thi s l e v e l of c e l l u l o s e , however, their 16 prote in conversion e f f i c i e n c y was not decreased. (Dupree and Sneed, 1966). An a d d i t i o n a l considerat ion with d ie tary f iber i s that i t increases the feca l volume, and hence lowers water q u a l i t y necess i tat ing greater aerat ion and/or water flow, and adds to environmental p o l l u t i o n (Bromley and Adkins, 1984). 2.9 Species Comparisons (Fish) Carbohydrate studies have been conducted on several species of f i sh that possess very d i f f erent d ie tary hab i t s . Despite the i r d i f f erent d ie tary adaptations, omnivorous, herbivorous and d e t r i t i v o r o u s f i s h a l l have high d ie tary prote in requirements, and the ir a b i l i t y to digest prote in i s comparable to that of carnivores (Tyt ler and Calow, 1985). Omnivorous f i sh are better able to u t i l i z e starch than carnivorous species . This was c l e a r l y demonstrated in a study by Furu ich i and Yone (1980) in which semi-puri f ied d ie t s containing 0 to 40% dextr in were fed to f i s h of three species; the carp, red sea bream, and y e l l o w t a i l (the former two species are omnivores and the l a t t e r i s a carn ivore ) . Growth was retarded and feed e f f i c i ency lowered in carp at 40%, sea bream at 30% and y e l l o w t a i l at 20% die tary d e x t r i n . As dextr in and prote in were well absorbed in a l l species regardless of dextr in l e v e l , poor u t i l i z a t i o n of dextr in was presumed. Takeuchi et al . (1979) reported that carp fed adequate prote in (32%) can e f f e c t i v e l y u t i l i z e carbohydrate 17 at a d ietary l eve l of 42% without adverse e f fects on growth, feed conversion or net prote in u t i l i z a t i o n (NPU). Feed e f f i c i e n c i e s of young carp fed a 45% a-starch d ie t and red sea bream fed a 30% a-starch diet have been reported to be 74% and 47%, respect ive ly (Furuichi and Yone, 1982b). In a study by Degani and Levanon (1987), the growth of eels was enhanced by increasing d ie tary glucose from 10% to 30%, with a prote in l e v e l f ixed at 45% of the d i e t . Increased carbohydrate also led to increased body f a t , ind ica t ing that i t was being used for energy and converted to fa t . It appears that the eel has a higher energy requirement and is better able to u t i l i z e d i f f e r e n t carbohydrate sources in comparison to the salmonid. Degani and V i o l a (1987) demonstrated higher spec i f i c growth rate , energy retent ion and feed conversion in European eels {Angui11 a angui I I a) fed d ie t s with 40% prote in and 38% wheat meal, as compared to those fed 50% and 20%, or 30% and 56%, re spec t ive ly . 2.9.1 S tra in Comparisons Chinook salmon possess great genetic d i v e r s i t y among many d i f f erent s t r a i n s , which is exhibi ted in wide v a r i a t i o n s in l i f e h i s tory patterns . "Ocean-type" chinook migrate to sea as underyearl ings, and "stream-type" af ter overwintering one year or more in fresh water. While most ocean-type chinook enter estuaries short ly af ter emergence as fry and subsequently spend up to several weeks there and 18 then migrate to the ocean, others spend 3 months in r i v e r s and reside very b r i e f l y in estuaries before ocean entry (Carl and Healey, 1984). Accompanying these di f ferences in migratory pat tern , are di f ferences in behaviour (Taylor and L a r k i n , 1986), growth and time of maturity (Withler et al., 1987). Comparison of s t ra ins or fami l ies for di f ferences in carbohydrate u t i l i z a t i o n have not previous ly been conducted on chinook salmon. However, such inves t igat ion has been c a r r i e d out in two studies on rainbow trout . Edwards et al. (1977) examined growth performances of f i n g e r l i n g rainbow trout from ten d i f f erent famil ies fed isonitrogenous d ie ts containing d i f f erent proportions of metabolizable energy (17% - 38%) as carbohydrate.^ Although there were s i g n i f i c a n t growth di f ferences between famil ies fed the same d i e t , there was no in terac t ion between diet and family , ind ica t ing un l ike ly prospects for genetic se lec t ion of enhanced carbohydrate u t i l i z a t i o n . In a companion study by Refs t ie and Austreng (1981), in which f ive rainbow trout fami l ies and f ive inbred groups were fed 15% - 49% of ME as carbohydrate, the prospects for se lec t ive breeding of s t ra ins for carbohydrate u t i l i z a t i o n were once again not very promising. Despite these negative f indings in rainbow trout , i t was postulated that chinook salmon may reveal some potent ia l for se lec t ion of t r a i t s associated with enhanced carbohydrate u t i l i z a t i o n owing to the aforementioned 19 di f ferences in the ir l i f e h i s tory and consequently in the d i v e r s i t y and amounts of natural prey ingested. 2.10 Carbohydrate Structure The primary carbohydrate encountered by salmonids in p r a c t i c a l cu l ture d ie t s i s complex carbohydrate in the form plant s tarch . Starch is composed of two types of glucosans c a l l e d amylose and amylopectin, the proportions of which vary depending on starch source. As examples, wheat, potato and ordinary corn starch are approximately 80% amylopectin and 20% amylose, while other starches may contain more than 50% of the l a t t e r . Amylose i s a l inear polymer of glucose with 0-1,4 glucoside l inkages , while amylopectin i s a branched glucose polymer bonded by a-1,4 and a-1,6 l inkages . Glycogen in l i v e r and muscle i s very s imi lar in s tructure to amylopectin, but with more extensive and shorter branches. (Linder, 1985; T i e t z , 1980) 2.11 Carbohydrate Digest ion and Absorption Diges t ib le carbohydrates contain mostly a-1,4 and a-1,6 l inkages , while f iber has a predominance of j3~1,4 bonds ( i e . c e l l u l o s e , pec t ins ) . During d iges t ion , the enzyme a-amylase hydrolyzes a-1,4, but not a-1,6, glucoside bonds. This leads to the formation of maltose and dextr ins . The l a t t e r are mixtures of d i f f erent s ize pieces of glucose polymer r e s u l t i n g from p a r t i a l h y d r o l y s i s . Some of these are subsequently hydrolyzed, however, res idual dextr ins remain 20 after amylopectin d iges t ion . Not only the composition, but also the structure ( i e . branching) of a s tarch af fects i t s hydrolys i s ra te . Studies of d igest ive enzymes in salmonids reveal the presence of a-amylase and disaccharidases , but the i r a c t i v i t i e s are low in comparison to those of herbivorous and omnivorous species (Nagayama and Sa i to , 1969). S u r p r i s i n g l y , a c t i v i t y of sucrase has been shown to exceed that of maltase in the trout (Buddington and H i l t o n , 1988), and th i s may also be true in chinook salmon (Buhler and Halver, 1961). The r e l a t i v e e f f i c i e n c i e s of absorption of carbohydrate are highest for monosaccharides, followed by disaccharides then cooked s tarch , and poorest in raw s tarch , in brook trout ( P h i l l i p s , 1948), and rainbow trout (Singh and Nose, 1967; Smith, 1971). 2.12 Carbohydrate Metabolism Salmonids exhibit prolonged hyperglycaemia when administered a glucose load, with plasma glucose remaining elevated 24 hours after the challenge (Palmer and Ryman, 1972; P h i l l i p s et al . , 1 948; Bergot, 1979b). This indicates that they have a poor a b i l i t y to u t i l i z e c i r c u l a t i n g glucose, a response s im i lar to that observed in d iabet i c mammals. It has been shown that the "aerobic oxidat ion and u t i l i z a t i o n of glucose in f i s h i s low, even when compensating for temperature differences" (Lin et al . , 1978; Cowey and Sargent, 1979). In studies using r a d i o - l a b e l l e d 21 glucose, L i n et al. (1978) working with coho salmon, and Bever et al. (1977) with kelp bass (Paralabrax sp.) found that the turnover rates of glucose were approximately 1/10 that in mammals (Katz et al . , 1976; Armstrong, 1979). Cowey et al. (1977a) ca lcu la ted the glucose space in trout to be 13.7% of body weight, while in the r a t , the determined value was 30% (Friedmann et al., 1965). Beamish et al. (1986) demonstrated that heat increment, as measured by the oxygen intake of i n d i v i d u a l f i s h in a swimming respirometer, was higher in rainbow trout fed a 30% cerelose d i e t , than in those fed an isonitrogenous 18% l i p i d d i e t . This i s consistent with the i n e f f i c i e n t u t i l i z a t i o n of glucose, and the resultant increased energy expenditure for i t s absorpt ion, metabolism and excre t ion . 2.12.1 G l y c o l y s i s and Gluconeogenesis G l y c o l y s i s i s the main pathway of carbohydrate degradation for energy and biosynthet ic reactions in most organisms (Fideu, 1983). Most of the enzymatic reactions of q l y c o l y s i s are revers ib le with the exception of three enzymes: hexokinase (HK); phosphofructokinase (PFK); and pyruvate kinase (PK) (Figure 1). When f i s h are producing the ir own glucose through gluconeogenesis, the g l y c o l y s i s pathway i s reversed. The steps that are i r r e v e r s i b l e (above) are bypassed by the corresponding enzymes, respec t ive ly : glucose-6-phosphatase; fructose diphosphatase; and pyruvate carboxylase (PC) in conjunction 22 Figure 1. Glucose Metabolism: g l y c o l y s i s and gluconeogenesis pathways. Glycogen Glucose UDP Glucose Hexoki nas ose-6-s phat as e T Glucose-1-phosphate Glucose-6-phosphate • — Gl ucos e-6-phos phat e dehydrogenase (G6PDH) PENTOSE PHOSPHATE PATHWAY Fructose-6-phosphate A. Phosphof r ucto-kinase (PFK) Fruct ose di phos phat as e (FD) Fructose-1-6-diphosphate I I I Phosphoenolpyruvate Pyruvat e Ki nas e (PK) Lactate < > Pyruvate Phosphoenolpyruvate j;ar boxyki nas e (PEPCK) Oxaloacetate Pyruvate carboxylase (PC) Acetyl CoA 23 with phosphoenolpyruvate-carboxykinase (PEPCK) (Figure 1). (Walton and Cowey, 1982) While g l y c o l y s i s occurs p r i m a r i l y in ske le ta l and heart muscle, gluconeogenesis i s c a r r i e d out mainly by the l i v e r . (Knox et al., 1980). Salmonids f u l f i l l the ir glucose needs p r i m a r i l y through gluconeogenesis, as the i r natural d ie t contains l i t t l e carbohydrate. Nonetheless, French et al. (1981) reported that "gluconeogenic a c t i v i t y in trout i s low compared with other f ishes and mammals". Before glucose can p a r t i c i p a t e in metabolism, i t must be phosphorylated to glucose-6-phosphate. The enzyme responsible i s hexokinase, a regulatory enzyme. HK a c t i v i t y has been shown to be much higher in muscle t i ssue than l i v e r t i ssue (Shibata, 1977). In the l i v e r s of many animals there i s an inducible hexokinase isoenzyme, glucokinase, spec i f i c for glucose, which has a high Km and is not i n h i b i t e d by the product glucose-6-P. With th i s enzyme, glucose loads can be qu ick ly handled, and blood glucose regulated. In f i s h , however, no isoenzyme with the propert ies of glucokinase has been detected (Nagayama and Ohshima, 1974, 1980; Cowey et al., 1977b), and hexokinase l eve l s are low in comparison to other g l y c o l y t i c enzymes. Glucokinase i s a lso absent in the carnivorous cat (MacDonald and Rogers, 1984). When compared to the r a t , f i s h have a lower capacity for phosphorylation of glucose in both muscle and l i v e r . For example, the a c t i v i t y of HK ( including glucokinase) in f i sh l i v e r i s 1/10 that in the rat l i v e r , and a c t i v i t y in the f i sh kidney is 24 1/3 that in the rat kidney (Cowey and Sargent, 1979). Cowey and Sargent (1979) conclude that th i s i s one of the primary reasons f i sh cannot metabolize glucose r a p i d l y . Once glucose i s converted to glucose-6-P i t can then enter into g l y c o l y s i s (Embden-Meyerhof pathway), glycogen synthesis , the pentose-phosphate pathway, or be reconverted to glucose. The two enzymes within g lyco lys i s 'which are regulatory in most animals are phosphofructokinase (PFK) and pyruvate kinase (PK) (Figure 1). The former is a complex enzyme responsible for the conversion of fructose 6-phosphate to fructose 1,6-diphosphate. The l a t t e r converts phosphoenolpyruvate to pyruvate. Pyruvate kinase was reported by Guderly and Cardenas (1980) not to be regulatory in the rainbow trout . PK has been found to be 10-fold higher than PFK in rainbow trout (Shibata, 1977). The pentose phosphate pathway i s another or secondary pathway of glucose catabol ism. The f i r s t step in th i s pathway is the dehydrogenation of glucose 6-phosphate by the enzyme glucose 6-phosphate dehydrogenase (G6PDH). It leads to the formation of NADPH and pentoses. NADPH i s an important reducing agent in biosynthesis of compounds such as fat ty ac ids , and pentoses, e spec ia l ly ribose 5-phosphate, used in nucle ic ac id biosynthesis (Leninger, 1982). However, the importance of th i s pathway in f i s h i s s t i l l not c l e a r . On the one hand i t has been reported to be of minor importance in glucose breakdown (Hochachka, 1961, 1969), while on the other, i t has been suggested to be the major 25 route for glucose catabolism in f i s h (Fideu et al., 1983). These l a t t e r invest igators concluded that due to very low PFK a c t i v i t i e s , and induction of increased G6PDH a c t i v i t y in trout on high carbohydrate d i e t s , the pentose phosphate path must be the main pathway of carbohydrate degradation. High l eve l s of G6PDH have been found in rainbow trout l i v e r s , and some invest igators have shown enhanced a c t i v i t y in f i s h fed high carbohydrate d ie ts (Hi l ton and Atkinson, 1982; Nagayama et al., 1973, 1975). 2.12.1.1 Enzyme Adaptation In carnivores , gluconeogenesis i s "more or less permanently switched on" and i s at i t s maximum in the absorptive s tate , whereas in omnivores, i t peaks hours af ter a meal in the postabsorptive s tate . This i s due to the fact that the carnivore has l i t t l e a b i l i t y to store carbohydrate. For example, the enzymes for amino ac id catabolism are r e l a t i v e l y non-adaptive to changes in d ietary prote in l eve l in the ca t , thus high nitrogen loss i s ob l igatory even i f d ie tary prote in is low (MacDonald and Rogers, 1984). Invest igat ions show that f i s h are s imi lar in th i s regard (Rumsey, 1981). Walton (1986) demonstrated that rainbow trout fed a high protein/ low carbohydrate (60%/l0%) diet versus a low prote in /h igh carbohydrate (20%/56%) diet exhibi ted s i g n i f i c a n t l y lower g l y c o l y t i c and higher gluconeogenic enzyme a c t i v i t i e s , however, most amino ac id ca tabo l i z ing enzymes invest igated were unaffected by d i e t . 26 Hi l ton and Atkinson (1982) found that adaptation v ia a l t e r a t i o n s in the a c t i v i t i e s of key g l y c o l y t i c and gluconeogenic enzymes occurred at carbohydrate l eve l s up to, but not above, 14% in rainbow trout fed isonitrogenous d i e t s . These invest igators also found that d ie tary carbohydrate d id not s i g n i f i c a n t l y af fect the percentage conversion of [ 1 ^C]alanine to glucose (Hi l ton and Atkinson, 1982). This poor adaptation of enzymes leads to a high obl igatory nitrogen los s , and thus, an i n a b i l i t y to conserve e s sent ia l amino acids when fed low prote in d i e t s . Omnivores and herbivores , on the other hand, are highly adaptive. 2.12.2 Glycogen Synthesis and Breakdown Liver glycogen in f i s h i s very s imi lar in structure to that found in mammals. In the synthesis of glycogen (Figure 2), glucose 6-phosphate i s converted to glucose 1-P, then to UDP-glucose and eventually the glucose units are attached onto the growing glycogen chain . This f i n a l step i s c a r r i e d out by glycogen synthase, an enzyme which ex is ts in an act ive ' a ' form (unphosphorylated) and inact ive 'b' form. A c t i v a t i o n i s brought about by a phosphatase, and i n a c t i v a t i o n by a prote in kinase, as i s found in mammals. In rainbow trout , the a c t i v i t y of glycogen synthase i s some 15-fold lower in white muscle than in the l i v e r and red muscle (Ingram, 1970). Glycogen breakdown occurs v ia a d i f f erent route than synthesis and i s regulated by the enzyme glycogen 27 Figure 2. L iver Glycogen Synthesis and Breakdown, Glycogen A Glyeogen Phosphor yl ase UDP Glucose UDP glucos e phos phor ylas e Glyeogen Synthas e Glucose-1-phosphate Phosphogl ucomutas e Glucose-6-phosphate Hexokinas e Glucose-6-phosphatase Glucose Enzymes ex is t in act ive and inact ive forms. Example of regulat ion: Glycogen sythetase kinase a c t i v a t i o n (ATP-->ADP) Hormonal > cAMP Glycogen synthetase regulat ion by I (act ive > inact ive) glucagon I adrenal in 4-Gl ycogen phoshphoryl ase kinase a c t i v a t i o n (ATP-->ADP) Glycogen phosphorylase ( inact ive > act ive) 2 8 phosphorylase, which a lso ex i s t s in act ive ' a ' form (phosphorylated in th i s case) , and less act ive 'b ' form. Regulation of these forms is a lso brought about by kinase (act ivat ion) and phosphatase (deactivation) enzymes. Phosphorylase breaks the a 1,4 g lycos id i c l i n k of glucose units in glycogen to release glucose 1-P, which i s subsequently converted to glucose 1-P. Glycogen phosphorylase enzymes are found in l i v e r and muscle but they d i f f e r in the ir s tructure and regu la t ion . In the l i v e r , where the purpose of glycogen breakdown i s to l i b e r a t e free glucose into the blood, an a d d i t i o n a l enzyme, glucose 6-phosphatase i s required to remove the phosphate group. The act ion of th i s enzyme i s i r r e v e r s i b l e . It has been found in the l i v e r s of several f i sh species , but ex i s t s in very low l eve l s in f i sh muscle (Nagayama et al., 1972, Shimeno and Ikeda, 1967) . While there i s evidence that these glycogen breakdown enzymes exis t in rainbow trout (Vernier and S i r e , 1978), phosphorylase appears to be absent in carp l i v e r (Murat, 1976), suggesting a d i f f erent pathway of glycogen breakdown in the l a t t e r species . Amylase has been found in the l i v e r s of carp and g o l d f i s h , but i s only ha l f as e f f ec t ive as phosphorylase in the breakdown of l i v e r glycogen in go ldf i sh (Murat, 1976; Picukans and Umminger, 1979). The importance of th i s enzyme in carbohydrate metabolism in f i s h i s controvers ia l (Christ iensen and Klungsoyr, 1987). 29 White muscle, which is the major t i ssue in f i shes , i s poorly vascular ized and thus r e l a t i v e l y anaerobic. Glycogen breakdown to lac tate i s c r u c i a l to maintain contract ion of the muscle. Gluconeogenesis from lactate (anaerobic g l y c o l y s i s ) and amino acids i s the main source of glucose for muscle glycogen (Moon, Walsh and Mommsen, 1985). L i v e r glycogen is general ly regarded as a read i ly access ib le glucose reserve. In rats i t f a l l s to very low leve l s within 24 hours and i s depleted in 1 to 2 days (Freedland, 1967). F i s h , by comparison, do not rap id ly remobil ize l i v e r glycogen (Cowey and Sargent, 1979), and there i s considerable in ter - spec ies v a r i a t i o n . The carp and Japanese ee l have been shown to maintain pre - fa s t ing concentrations of l i v e r glycogen af ter 20 days without food (Larsson and Lewander, 1973). In fac t , the carp can be starved more than 100 days without deplet ing i t s l i v e r glycogen (Nagia and Ikeda, 1971). In rainbow trout , l i v e r glycogen decl ined by 80% after 20 days of feed withdrawal, then increased to a constant l e v e l with continued fa s t ing , during a 60 day experimental per iod . However, when phys ica l stress was imposed, l i v e r glycogen dropped at a much higher rate: a 40% decrease occurred in 30 minutes then new synthesis qu ick ly replenished i t in 45 minutes (Morata et al . , 1982). 30 2.12.2.1 L iver Glycogen Studies Feeding high carbohydrate d ie t s to f i s h can lead to substant ia l e levat ion in hepatic glycogen l e v e l s . L iver glycogen accumulation has been reported in salmonids fed d ie t s supplemented with glucose, dextr in and ( less so) other sugars and starches (Hi l ton and Atkinson, 1982; H i l t o n et al . , 1982; Bergot, 1979a; Palmer and Ryman, 1972; Buhler and Halver , 1961; P h i l l i p s et al., 1948). Palmer and Ryman (1972) a lso found that go ldf i sh fed 25% white maize dextr in for 51 days exhibi ted hyperglycaemia, elevated glycogen, deplet ion of prote in and deposits of fat in t h e i r l i v e r s . The e f fects of elevated l i v e r glycogen l eve l s are large ly unknown. Several reports indicate that d ie tary d i g e s t i b l e carbohydrate l eve l s above 20% leads to excess l i v e r glycogen and impairment of l i v e r function in trout (Dixon and H i l t o n , 1981; H i l t o n and Atkinson, 1982; H i l t o n , 1982). P h i l l i p s et al. (1948) reported abdominal swel l ing , hepatomegaly, and increased morta l i ty in brook trout fed only 12 % die tary d i g e s t i b l e carbohydrate. In other inves t igat ions higher morta l i ty rates have not accompanied the increase in l i v e r glycogen (Refstie and Austreng, 1981; Bergot, 1979; Buhler and Halver, 1961). However, poss ible sublethal e f fects could be detrimental over a longer term (Dixon and H i l t o n , 1981, 1985). H i l t o n (1982) demonstrated that pre - s tarvat ion diet and water temperature both af fect l i v e r glycogen l e v e l and l i v e r weight during s tarvat ion in rainbow t r o u t . In th i s 31 inves t iga t ion , l i v e r glycogen and l i v e r weight increased with increas ing dietary cerelose l e v e l (0 to 34%) and decreasing temperature. L iver glycogen l e v e l s , which had reached over 10 %, and l i v e r weight both decl ined to contro l l eve l s between 7 and 12 days after feed withdrawal. 2.13 Endocrine Control 2.13.1 Insu l in Insul in i s a hormone involved in anabolism, d i r e c t i n g energy storage from ingested food energy. Its anabolic functions include: per iphera l glucose u t i l i z a t i o n , storage of glucose in glycogen or s h i f t of glucose into g l y c o l y s i s , enhanced fa t ty ac id uptake in adipose, increased l ipogenes i s , and increased uptake of aqjino acids and the i r deposit ion into prote ins . Insul in targets the l i v e r , muscle and adipose t i s sue . In f i s h , i t s primary s tructure i s very s imi lar to that in mammals (Christ iensen and Klungsoyr, 1987). As in mammals, i n s u l i n has been shown to i n h i b i t gluconeogenesis in f i s h (Cowey et al., 1977). Its e f fect on glycogen deposit ion in f i s h i s not c l e a r , and in some s tudies , t i s sue glycogen has ac tua l ly decreased after i n s u l i n in jec t ion (Ince, 1983). It appears to increase the clearance of glucose v ia oxidation rather than glycogen deposit ion (Ince, 1983). F i s h d i f f e r from mammals in the i r a b i l i t y to metabolize carbohydrates p a r t i c u l a r l y with respect to endocrine c o n t r o l . Many invest igators have ascribed the poor 32 u t i l i z a t i o n of d ie tary glucose by f i s h to an insuf f i c i ency of i n s u l i n and consider f i s h to resemble Type-I d iabet i c s due to i n s u l i n def ic iency (Palmer and Ryman, 1972; Cowey et al . , 1977a, 1977b; Furu ich i and Yone, 1981). Insu l in has been shown to exert some contro l over blood glucose in f i s h . Fast ing hyperglycaemia has been observed in f i s h fol lowing removal of the pancreas or i s l e t s , or treatment of the i s l e t s with cytotoxins (Matty, 1985). Inject ion of exogenous mammalian or p i sc ine i n s u l i n causes a hypoglycaemic response in f i s h , and the effect i s more pronounced when f i sh i n s u l i n i s used (Ince, 1983). In a 1972 study by Palmer and Ryman, i n t r a c a r d i a l administrat ion of i n s u l i n B . P . resul ted in marked hypoglycaemia in rainbow trout , and adminis trat ion of i n s u l i n simultaneous with glucose loading improved glucose tolerance (Palmer and Ryman, 1972). Work by these authors also suggests that the mechanism of act ion of i n s u l i n s t imulat ion is d i f f eren t in f i s h than in mammals (Palmer and Ryman, 1972). St imulat ion of i n s u l i n in f i s h i s brought about by d i f f erent metabolites than in mammals. Thorpe (1976) showed c o r r e l a t i o n s between amino ac id and i n s u l i n l eve l s in cod and rainbow trout , but found no ef fect of glucose on i n s u l i n secre t ion . In 1977, Ince and Thorpe compared ef fects of various amino acids on in vitro i n s u l i n secret ion in perfused European eel pancreas and found that a l l amino acids tested were more e f f ec t ive than glucose. In vitro studies using toadf ish i s l e t t i ssue have shown that high 33 l eve l s of glucose st imulate i n s u l i n release (Tashima and C a h i l l , 1968). However, the fact that glucose adminis trat ion leads to hyperglycaemia indicates a l imi ted in vivo capacity for i t s re lease . Hi l ton and Atkinson (1982) demonstrated that feeding rainbow trout a carbohydrate-r ich d ie t for several months d id not af fect the s ize or number of i n s u l i n secret ing /3-cells in the pancreas. In toadf i sh , glucose was found to act s y n e r g i s t i c a l l y on leucine st imulat ion of i n s u l i n secret ion (Patent and Foa, 1971). Huth and Rapoport (1982), however, found no st imulatory effect by leucine or glucose on i n s u l i n production in carp. Ince and Thorpe (1977) demonstrated that glucose doses exceeding 100 mg/kg body weight do not further stimulate the secret ion of i n s u l i n in the s i l v e r ee l (Angui11 a angui 11 a). It has been suggested that glucose may function only to maintain basel ine i n s u l i n l eve l s in f i s h , while amino acids are the main regulators of i n s u l i n production and secret ion (Christ iansen and Klungsoyr, 1987). In contrast , H i l t o n et al . (1987b) more recently found that plasma i n s u l i n was s i g n i f i c a n t l y higher in rainbow trout fed a high-carbohydrate (25% glucose) versus a low-carbohydrate d i e t , ind ica t ing that glucose st imulates i n s u l i n secret ion in t r o u t . These authors suggested that the trout are not i n s u l i n - d e f i c i e n t and they may be more s imi lar to type-II (non- insul in dependent) d iabet i c s (Hi l ton et al., 1987b). 34 Various techniques have been developed to measure i n s u l i n l eve l s in mammals and f i s h . Many studies have been conducted using radioimmunoassays (RIAs). In th i s method antibodies against i n s u l i n , produced in a host animal and subsequently r a d i o l a b e l l e d , are used to detect plasma i n s u l i n in vitro. I n i t i a l l y , in the RIAs developed for measuring c i r c u l a t i n g hormone l eve l s in f i s h , ant i sera to mammalian i n s u l i n was used. This d id not give accurate quant i ta t ive resul t s (Pl isetskaya et al., 1976). Subsequently, i t has been found that the i n s u l i n of salmon crossreacts weakly with ant i sera to mammalian i n s u l i n commonly used in RIAs, and bovine i n s u l i n has no crossreact ion with ant i sera to f i s h i n s u l i n (Pl isetskaya et al., 1985). The use of f i s h i n s u l i n of the same or even d i s t a n t l y re lated species has improved the assays cons iderably . Results may be several fo ld higher when a homologous RIA is used (Pl isetskaya et al., 1986). Resting plasma i n s u l i n l eve l s in salmon have been found to be in the area of 4-6 ng/ml, which is much higher than those of mammals. High c i r c u l a t i n g plasma i n s u l i n leve ls have a lso been found in other f i s h species . Muggeo et al. (1979) suggested that the higher c i r c u l a t i n g i n s u l i n l e v e l in f i s h i s probably a compensation for i t s lower potency. (Pl isetskaya et al., 1986). Temperature i s l i k e l y to a f fec t the function of i n s u l i n , for example, p r o i n s u l i n conversion to i n s u l i n is very slow at low temperatures. 3 5 2.13.2 Glucagon In mammals, glucagon functions to increase blood sugar p r i m a r i l y v ia breakdown of l i v e r glycogen, and also by s t imulat ion of gluconeogenesis. In f i s h the hormone exerts i t s ef fect p r i m a r i l y through st imulat ion of gluconeogenesis (Hayashi and Ooshiro, 1985; T y t l e r and Calow, 1985). 2.13.3 Thyroid Hormone Thyroid hormone has a ca lor igen ic e f fect in mammals, as demonstrated by oxygen consumption s tudies . In f i s h , i t s e f fects on oxygen consumption are unclear (Tyt ler and Calow, 1985). H i l t o n et al.(1987b) found that supplementation of 3 , 5 , 3 ' - t r i i o d o - L - t h y r o n i n e (T3) d id not enhance the u t i l i z a t i o n of glucose in rainbow trout and had no s i g n i f i c a n t effect on plasma i n s u l i n l e v e l s . 2.14 Oral Glucose Tolerance Glucose tolerance is determined by the "rate at which inherent mechanisms for removing excess glucose from the blood perform the ir functions" (Linder, 1984). The glucose tolerance tes t , or GTT, i s a common diagnost ic test used for the detect ion of diabetes or other disorders of glucose metabolism. In t h i s t e s t , the subject 's blood glucose i s monitored subsequent to administrat ion of a large dose of glucose. In tes t ing humans, the usual procedure is to administer a 100 g dose of glucose o r a l l y fol lowing an 36 overnight fas t , then measure blood glucose at spec i f i c in t erva l s afterward. The shape of the re su l t ing blood glucose curve ( i e . height and time of occurrence of blood glucose peak) i s determined by several fac tors . These include: the secret ion of s u f f i c i e n t i n s u l i n ; the effect iveness of i n s u l i n ; the presence of other factors involved in the act ion and binding of i n s u l i n ; the rate of i n s u l i n breakdown; the presence of i n s u l i n antagonists; and secret ion of counterregulatory substances, such as glucagon, which stop the f a l l in blood glucose after i n s u l i n has acted (Linder, 1984). In human subjects , a blood glucose l e v e l higher than 160 mg/dl at 30 to 60 minutes post GTT i s abnormal. Return to the res t ing glucose l e v e l of 70 - 105 mg/dl normally takes 1 to 2 hours to occur. Glucose intolerance and diabetes are indicated by elevated res t ing blood glucose l e v e l s , a higher than normal and/or delayed peak and a delayed return to normal. In humans, i f blood glucose exceeds 180 mg/dl , the kidney's resorpt ion capacity i s surpassed, and glucose i s lost in the urine (Linder, 1984). Other tests such as the i n s u l i n tolerance test may be conducted to evaluate ind iv idua l s that are res i s tant to i n s u l i n , or have other endocrine d i sorders . Many invest igators have reported glucose intolerance in f i s h equating i t with type I diabetes in humans. Palmer and Ryman (1972) conducted o r a l glucose tolerance tests on year l ing rainbow trout . Pers i s tent hyperglycaemia resu l ted , 37 with blood glucose r i s i n g from i t s fas t ing l e v e l of approximately 80 mg/dl to over 500 mg/dl in 7 hours, and remaining s l i g h t l y elevated at 24 hours. Simultaneous adminis trat ion of i n s u l i n with glucose improved tolerance s i g n i f i c a n t l y . In teres t ing ly , prespawning females exhibited a markedly improved ora l glucose to lerance . Wilson and Poe (1987) examined o r a l g lucose'tolerance in channel c a t f i s h using several sugars. While glucose and maltose produced the most hyperglycaemic response, administrat ion of dextr in led to a more gradual r i s e in plasma glucose due to i t s slower d iges t ion/absorpt ion rate . The authors suggested that th i s allows the absorbed glucose to synchronize with the peak i n s u l i n secret ion and thus expedite the u t i l i z a t i o n of the c i r c u l a t i n g glucose. Response curves for sucrose and fructose were delayed due to the poor absorption of fructose and i t s lack of conversion to glucose. These resu l t s indicate that channel c a t f i s h , although better able to to lerate carbohydrate then salmonids, s t i l l do not use monosaccharides and disaccharides well as energy sources. Furu ich i and Yone (1981) measured plasma i n s u l i n l eve l s during GTT in carp, red sea bream and y e l l o w t a i l , and found that they peaked 2 hours after the o r a l glucose chal lenge, p a r a l l e l i n g plasma glucose l e v e l s . They suggested that the i n s u l i n pattern was very s imi lar to that of a d iabet ic human. Further inves t igat ion by these workers (Furuichi and Yone, 1982a, 1982c) led them to conclude that i n s u l i n 38 i n s u f f i c i e n c y was the cause of poor carbohydrate u t i l i z a t i o n . Furu ich i and Yone (1982b) conducted carbohydrate tolerance tests in red sea bream using glucose, dextr in or a - s tarch . At two hours af ter adminis trat ion , the point at which i n s u l i n secret ion has been shown to peak (Momose et al., 1963), 95% of the glucose, 65% of the dextr in and only 4% of the s tarch had been absorbed. Most of the starch was absorbed during the next 3 to 10 hours after adminis trat ion; peak absorption occurring at 5 to 7 hours. In the starch group, blood sugar peaked before the point of maximum absorpt ion. It was suggested that the delayed absorption of s tarch , occurring after i n s u l i n secre t ion , allowed the s tarch to be well u t i l i z e d (Furuichi and Yone, 1982b). 39 3 GENERAL MATERIALS AND METHODS 3.1 His tory and Maintenance of Experimental F i s h Between March 23 and 30, 1987, approximately 500 f i sh of each of 10 s tra ins of B . C . chinook salmon fry {Oneorhynchus tshawytscha) were brought to the Department of F i sher i e s and Oceans West Vancouver Laboratory in West Vancouver. The stocks included s ix coastal chinook s t r a i n s ; Capi lano, Big Qualicum, Quinsam, Robertson Creek, N i t i n a t and Harr i son , and four inland chinook s t r a i n s ; Quesnel, Shuswap, Eagle River and Clearwater. Upon a r r i v a l , the f i s h weighed between 1 and 3 grams, the Quesnel stock averaging s l i g h t l y higher (approx. 2 to 4 grams). Each stock was held in a 1100 1 oval f iberg lass tank covered with a nylon mesh l i d , and supplied with fresh well water ( 1 1 ° C ) at an approximate flow rate of 15-20 1/min. Oregon moist p e l l e t s were fed to sa t ia t ion 3 times d a i l y , i n i t i a l l y , then the feeding•frequency was reduced to 2 times d a i l y in September. A l l stocks were dip-vaccinated against Vibriosis (ordalii and angui 11 arum) at approximately 8 grams body weight. To determine readiness for s m o l t i f i c a t i o n , the f i s h were sampled for weight. A l s o , they were observed for degree of s i l v e r i n g and jumping behaviour. On June 12, 1987, sea water transfer was i n i t i a t e d in the Quinsam, Capi lano, Harr i son , Robertson Creek, N i t i n a t and Quesnel s t r a i n s . The average weight of these f i s h ranged from 6.5 to 7.0 g, except for the Quesnel stock, which averaged 9.5 4 0 g. The Qualicum and Shuswap stocks were s tarted on June 26, when the ir average weights approached 7.0 g, and Eagle River and Clearwater on July 23, when the f i sh weighed an average of 6.5 and 5.5 g, re spec t ive ly . The f i sh were gradually sh i f ted from fresh water (FW) to sea water (SW) over a period of 12 to 15 days. The sea water was pumped in from Burrard Inlet at a depth of 30 m, and the June-July temperature was 10 to 1 0 . 5 ° C . The inland s t r a i n s , not normally accustomed to early marine entry , exhibited varying degrees of d i f f i c u l t y in adapting to seawater. Clearwater chinook f a i l e d to smoltify which resul ted in severe morta l i ty and s tunt ing . It was discovered sometime (months) l a t e r that many of the Eagle River and Shuswap f i s h had also f a i l e d to properly smolt i fy , r e s u l t i n g in growth reduct ion, some stunt ing, parr reversion and increased disease. Experiments were delayed, unfortunately , u n t i l the fol lowing spring (1988), when new laboratory f a c i l i t i e s became ava i lab l e for experimentation. The stocks were p e r i o d i c a l l y sampled for weight, c u l l e d to some extent, and div ided among a few a d d i t i o n a l tanks where poss ib l e . Feeding was reduced to l i m i t growth during part of the f a l l and winter due to r e s t r i c t e d tank space. Sea water temperature reached 13°C in September and dropped to about 7°C in February 1988. Dissolved oxygen (DO) values varied between 6 and 8 ppt, and were lowest after feeding. S a l i n i t y ranged from 21 to 30 ppt . L ight ing followed the 41 natural photoperiod. The chinook stocks were treated once with oxytetracyc l ine hydrochloride and once with sulphamerazine due to outbreaks of Vibriosis. In ear ly February of 1988 the nine remaining stocks were moved to 3.0 m diameter 7500 1) outdoor c i r c u l a r tanks with a flow rates of approximately 35 1/min. On March 16, 1988, these stocks were moved to the new DFO f a c i l i t y into 2.5 m diameter (<=* 6000 1) outdoor c i r c u l a r tanks, on the same seawater system, where they remained u n t i l experimentation. Short ly after th i s time a l l f i s h were sh i f ted from the OMP die t (3.0 mm) to a commercial dry diet (4.0 mm). By the end of A p r i l , the s t r a i n average weights var ied from 53 g (Harrison) to 92 g (Quesnel). 4 2 4 EXPERIMENT 1. The Ef fec ts of Feeding a High Versus Low Carbohydrate Diet on Growth, Body Composition, Feed and Prote in U t i l i z a t i o n , L iver Glycogen Concentration and L i v e r Weight in Selected Stra ins of B. C. Chinook Salmon. 4.1 MATERIALS AND METHODS 4.1.2 Diet Preparation and Composition The compositions of the two test d i e t s , re ferred to as "control" and "high carbohydrate" d i e t s , are given in Table 1. The test d iets were formulated to be isonitrogenous (440 g prote in /kg diet ) and i s o c a l o r i c (3633 kcal ME/kg) (see Table 2). Diets were pe l l e ted under reduced steam pressure in a C a l i f o r n i a Pe l l e t M i l l through a 5/32" die then a i r - d r i e d . As the herr ing o i l content of the contro l d ie t would have been too high for p e l l e t i n g , a port ion of the o i l was excluded from the mash. After p e l l e t i n g , the remaining o i l was sprayed onto the contro l d ie t in a ro ta t ing drum. The d ie ts were stored in p l a s t i c bags inside paper bags and kept frozen at - 1 8 ° C . Table 1 - Composition of test d ie t s as fed. 43 LC D i e t 1 HC Diet (g/kg) (g/kg) Steam-dried herr ing meal 387.86 387.86 Freeze-dr ied euphausids^ 155.29 155.29 Poultry-by-product meal 120.83 120.83 a -ce l lu lose 185.71 -P r e - g e l a t i n i z e d wheat starch - 286.97 Herring o i l ( s tab i l i zed) 101.26 -Vitamin supplement 4 1 4.35 14.35 Mineral supplement^ 9.57 9.57 Permapell 1 3.66 13.66 Choline ch lor ide (60%) 4.78 4.78 Ascorbic ac id 1.91 1 .91 Chromic oxide 4.78 4.78 1000.00 1000.00 LC: low carbohydrate/high l i p i d (control) d i e t . HC: high carbohydrate/low l i p i d d i e t . S t a b i l i z e d with 200 ppm ethoxyquin Vitamin supplement supplied the fol lowing l eve l s of nutr ients per kg of d ie t as fed: D-calcium pantothenate, 185.2 mg; pyridoxine H C l , 43.0 mg; r i b o f l a v i n , 57.4 mg; n i a c i n , 286.8 mg; f o l i c a c i d , 19.1 mg; thiamin mononitrate, 38.8 mg; b i o t i n , 2.87 mg; cyanocobalamine, 57 ug; menadione 25 mg; d l -a - tocophery l acetate, 573.6 IU; c h o l e c a l c i f e r o l , 2,294.4 IU; r e t i n o l acetate, 9,560 .0 IU; i n o s i t o l , 382.4 mg. Mineral supplement supplied the fol lowing l eve l s of nutr ients in mg/kg of diet as fed: cobalt (as C o C l 2 * 6 H 2 0 ) , 0.96; copper (as C u S 0 4 « 5 H 2 0 ) , 3.29; iron (as F e S 0 4 « 7 H 2 0 ) , 40.2; magnesium (as M g S 0 4 « 7 H 2 0 ) , 371; manganese (as M n S 0 4 « H 2 0 ) , 84.9; selenium (as N a 2 S e 0 3 ) , 0.096; zinc (as ZnS0 4>7H 20), 65.7; iodine (as K I 0 3 ) , 4.8; f luor ine (as NaF), 4.3. 44 Table 2 - Calculated metabolizable energy values of test d ie ts on a moisture-free bas i s . Energy Source kcal ME per kg LC Diet 1 HC D i e t 1 g/kg kcal ME per kg g/kg kcal ME per kg Prote in 4.5 2 440.0 1980 440.0 1980 L i p i d 8.5 2 187.5 1594 81 .64 694 CHO-animal 3.8 2 15.6 59 15.6 59 -ge la t . starch 3.0 3 — - 300.0 900 Tota l ME 3633 3633 LC Diet = low carbohydrate (control) d i e t ; HC Diet = high carbohydrate d i e t . From Beamish et al . (1986). Assumed 75% d i g e s t i b i l i t y at th i s d ie tary inc lus ion l e v e l (Singh and Nose, 1967). 45 4.1.3 F i s h Select ion and Tank A l l o c a t i o n Two coastal chinook s t r a i n s , Robertson Creek and Big Qualicum, and two inland chinook s t r a i n s , Quesnel and Shuswap, were selected for th i s study. On May 28, 1988, 50 f i s h were randomly selected as a representative sample from each of these s t r a i n s , anesthetized in 2-phenoxyethanol (0.35 ml/1) and weighed to the nearest 0.1 g. Due to di f ferences in growth rates , the s t a r t i n g weights and weight d i s t r i b u t i o n s were not homogeneous between s t r a i n s . The t o t a l number of experimental f i s h selected from each s t r a i n was as fol lows; 120 Robertson Creek, 120 Big Qualicum, 105 Quesnel, and 96 Shuswap chinook (there were fewer f i s h of the l a t t e r two s t r a i n s ) . Each stock was randomly d iv ided into 4 equal groups placed in separate 800 1 indoor f iberg lass tanks. Therefore, out of a t o t a l of 16 experimental tanks, there were 4 tanks of 30 Robertson Creek, 4 tanks of 30 Big Qualicum, 4 tanks of 26 (note: 1 tank had 27) Quesnel, and 4 tanks of 24 (note: 1 tank had 23) Shuswap chinook. I n i t i a l dens i t i es ranged from 4.0 kg/m 3 to 4.5 kg/m 3 . Each tank was provided with aerat ion and a seawater flow rate of 10 1/min. Water temperature ranged from 10 °C to 13 °C over the course of the experiment. Light ing followed the natural photoperiod. The 16 experimental tanks were s i tuated in 2 rows of 8, with the four groups of chinook from each s t r a i n randomly arranged. 46 Experimental f i s h were acclimated for 20 days, during which time they were gradual ly sh i f ted from the commercial dry diet to a contro l d i e t , over a 7 day per iod . 4.1.4 Start of Experiment Each experimental sampling was c a r r i e d out on i n d i v i d u a l rows, over two consecutive days. Feed was withheld 1 day p r i o r to weighing the f i s h . On June 17, 1988 (Day 0 of experiment), each group of f i s h from row 1 was captured and div ided among three 30 1 p l a s t i c buckets of aerated seawater. The f i r s t six f i s h from each tank were s ingly k i l l e d by an overdose of anesthetic (0.8 ml/1 2-phenoxyethanol), weighed (to the nearest 0.01 g) and measured (to the nearest 0.1 cm). Four f i s h had t h e i r l i v e r s rap id ly removed, weighed (± 0.0001 g) , f lash frozen in l i q u i d ni trogen, and stored at - 4 0 ° C for la ter glycogen determination. A general health check was then c a r r i e d out on each carcass , examining major organs, e spec ia l l y the kidney, for any evidence of disease. Two f i s h were frozen whole on dry ice then stored at - 1 8 ° C for subsequent proximate a n a l y s i s . The remaining f i sh were anesthetized (0.35 2-phenoxyethanol), weighed and measured. On June, 18, the fol lowing day, the second row of experimental groups was sampled in the same manner. As a l l f i s h had been fed the contro l d ie t to th i s po int , samples from two pa irs of tanks containing the same chinook s t r a i n , were pooled. 47 Experimental feeding commenced on June 18 for row 1 and June 19 for row 2. Two of the four experimental groups of chinook within each s t r a i n were continued on the contro l (LC) d iet and the other two were fed the high carbohydrate (HC) d i e t . A l l f i sh were fed twice d a i l y to s a t i a t i o n , from 9:00 to 10:00 am and from 3:00 to 4:00 pm. Da i ly feed intake was recorded for each group. Feed was withheld one day p r i o r to sampling. F i sh weights and lengths were again recorded on day 21 (July 7 and 8, rows 1 and 2 respect ive ly) and day 42 (July 28 and 29) of the experiment. On day 63 (August 18 and 19), the f i n a l day of the growth t r i a l , the f i r s t 12 f i s h from each tank were k i l l e d . Eight f i s h had the ir l i v e r s removed and quick-frozen in l i q u i d ni trogen, 4 f i sh were frozen whole on dry ice for proximate a n a l y s i s , and the remaining f i s h were weighed and measured, as prev ious ly . For the ensuing 21 days, feed was withheld from a l l groups of remaining f i s h . On Sept 8 and 9, the f i r s t 8 f i s h from each tank were s a c r i f i c e d for l i v e r removal, as before. A l l f i s h weights and lengths were recorded. 4.1.5 Proximate Body Composition This analys is consisted of dry matter, crude p r o t e i n , crude l i p i d and ash determinations of the f i s h carcasses from each treatment group on days 0 and 63 of the growth t r i a l . The frozen whole chinook had been stored at - 1 8 ° C for approximately 4 months. Each sample, cons i s t ing of 4 48 f i s h , was chopped i n t o p i e c e s while f r o z e n . Feed was removed from the stomach and i n t e s t i n e , so i t would not a f f e c t the r e s u l t s . The fro z e n s e c t i o n s were ground through a meat g r i n d e r , 2 or 3 times. The r e s u l t i n g p a s t e - l i k e mixture was pressed i n t o a 1 cm t h i c k p a t t y and r e f r o z e n , then f r e e z e - d r i e d f o r 72 hours. Each f r e e z e - d r i e d sample was reground to a f i n e powder with a Braun c o f f e e g r i n d e r . A l l a n a l y s e s were performed i n t r i p l i c a t e . Dry matter was determined a f t e r d r y i n g the powdered samples f o r 24 hours at 85°C. L i p i d content was determined by a m o d i f i c a t i o n of the B l i g h and Dyer (1959) e x t r a c t i o n method. Approximately 2.0 g of f r e e z e - d r i e d ground f i s h was mixed fo r three minutes i n a blender with 10 ml c h l o r o f o r m , 20 ml methanol and 10 ml water. Another 10 ml of c h l o r o f o r m was added to the mixture, f o l l o w e d by 30 seconds of b l e n d i n g . F i n a l l y , 1/2 teaspoon of f i l t e r - a i d and approximately 5 ml water were added, f o l l o w e d by another 30 seconds of b l e n d i n g . The mixture was passed through a s i n t e r e d g l a s s funnel i n t o a vacuum f l a s k . A f t e r f i l t e r i n g , the contents of the vacuum f l a s k were emptied i n t o a 50 ml g l a s s graduated c y l i n d e r . The f l a s k was then r i n s e d with a small amount of 1:1 chloroform:methanol s o l u t i o n and the washings added to the graduated c y l i n d e r . The s o l v e n t l a y e r s were allowed to separate o v e r n i g h t . The volume of the c h l o r o f o r m / l i p i d l a y e r was recorded and the methanol l a y e r was then removed by s u c t i o n . T r i p l i c a t e 5 ml subsamples of the 49 c h l o r o f o r m / l i p i d layer were evaporated and dr i ed in t i n s to determine l i p i d weight. Tota l % carcass l i p i d was then c a l c u l a t e d . Protein content was determined by the micro-Kje ldahl technique using a Technicon Autoanalyzer. A 0.1 to 0.15 a l iquot of each sample was digested for 1 hour in 10 ml of concentrated H2SO4 with a mercury cata lys t and 2.0 ml hydrogen.peroxide. After coo l ing , d i s t i l l e d water was added to the mixture, bringing the t o t a l volume to 50 ml . Samples were then run through the autoanalyzer, along with blanks and nitrogen standards. Determination of nitrogen by th i s procedure i s based on a co lor imetr i c method in which a green co lor i s formed by the react ion of ammonia sodium s a l i c y l a t e , sodium ni tropruss ide and sodium hypochlori te in a buffered a l k a l i n e medium (pH 12.8 - 13.0). The ammonia s a l i c y l a t e complex is read at 660 nm wavelength in the colorimeter chamber. Protein was ca lcu la ted from sample absorbance peaks. Ash content was determined by plac ing the samples in ceramic cruc ib l e s in a muffle furnace at 600°C for 4 hours, then weighing the remaining res idue. 4.1.6 L iver Glycogen Determination Liver glycogen was quant i f i ed by a modif icat ion of the Hassid and Abraham (1957) anthrone procedure. A 5.0 g piece of frozen l i v e r was quick ly sectioned and mixed with 3.0 mis of 30% (w/v) potassium hydroxide. After 20 minutes of 50 digest ion in a b o i l i n g water bath, 5.0 mis of ethanol was added to p r e c i p i t a t e the glycogen. Samples were then vortexed, centrifuged at 10000 rpm for 10 minutes, and the supernatants d iscarded. D i s t i l l e d water was added to d i s so lve the glycogen. A 2.0 ml a l iquot of the so lut ion was slowly added to 4.0 mis of fresh anthrone reagent, cons i s t ing of 0.2% anthrone in 98% sulphuric a c i d . The mixture was vortexed and heated for 10 minutes in a b o i l i n g water bath, then cooled. In th i s step, glycogen was hydrolyzed by the ac id and l ibera ted glucose reacted with anthrone, al lowing co lor imetr i c determination. Absorbance was read in a Unicam SP1800 u l t r a v i o l e t Spectrophotometer at a 620 nm wavelength. Glucose content was ca l cu la ted from a standard/absorbance curve generated using a set of 5 glucose standards, ranging in concentration from 5 to 25 ug/ml. 4.1.7 S t a t i s t i c a l Analyses Data were analyzed using the SAS analys i s of variance procedure (SAS version 6.03, 1988). Each set of 4 experimental units within a chinook s t r a i n was analyzed separately by one-way analys i s of variance , with diet as the treatment or c lass v a r i a b l e . The experimental model (for each s tra in ) was a completely randomized design. Data from Shuswap chinook were not included in analyses due to serious disease outbreak in a l l experimental groups of th i s s t r a i n . 51 4.2 RESULTS 4.2.1 Proximate Analyses of Diets and Prote in Sources The proximate compositions of d iet 1 (control or low carbohydrate) and diet 2 (high carbohydrate) , and the 3 prote in sources included in these d ie ts (steam-dried herring meal, f reeze-dr ied euphausids and poultry-by-product meal) are given in Tables 3 and 4, r e spec t ive ly . The proximate ana lys i s resu l t s of d ie t s 1 and 2 varied only s l i g h t l y from expected values for prote in (44.0% of d iet ) and l i p i d (18.75% of d iet 1 and 8.16% of d iet 2). 4.2.2 Growth and Condit ion Factor Figure 3 i l l u s t r a t e s the growth of the Quesnel, Big Qualicum and Robertson Creek chinook over the nine week feeding per iod . Average weights are also shown in Table 5. Data from the Shuswap s t r a i n were excluded from a l l experimental analyses due to a high incidence of BKD (bac ter ia l kidney disease) in the stock, which became apparent during the study. Upon commencement of the growth t r i a l , the four chinook s tra ins examined d i f f e r e d s i g n i f i c a n t l y in mean body weights, and variances around the means (see Table 5). Therefore d i r e c t s t r a i n comparisons could not be made, and analys i s was r e s t r i c t e d to the e f fects of d iet within i n d i v i d u a l s t r a i n s . 52 Table 3 - Proximate analyses of d i e t s . Diet Treatment 1 % D.M. % C P 2 % C L 3 % Ash 1 LC 92.14 46.16 19.47 10.98 2 HC 92.10 46.34 9.1 1 1 1 .00 1 LC = low carbohydrate/high l i p i d (control) d i e t ; HC = high carbohydrate/low l i p i d d i e t . 2 Crude prote in was estimated from nitrogen (N) determination, using the macro-Kjeldahl technique (CP = %N X 6.25). 3 Crude l i p i d was determined by the B l igh and Dyer (1959) extract ion procedure. Note: Pro te in , l i p i d and ash expressed on a dry matter ba s i s. 53 Table 4 - Proximate analyses of p r o t e i n sources f o r experimental d i e t s (dry matter b a s i s ) . F e e d s t u f f %DM1 %CP 2 %CL 3 %Ash Steam-dried H e r r i n g Meal 93.4 71.2 10.2 16.0 F r e e z e - d r i e d Euphausids 92.4 61 .0 14.6 13.6 P o u l t r y By-product 95.0 65.7 17.7 12.5 Dry matter. Crude p r o t e i n content determined by the m i c r o - K j e l d a h l technique. Crude l i p i d content determined by the B l i g h and Dyer e x t r a c t i o n method (1959). Figure 3 : Growth of Chinook Salmon of Three Strains Fed Low VS High Carbohydrate Diets Over a 63 Day Period. Time (days) Means ± 1 standard error of the mean, N=2. 55 Table 5 - Mean body weights of treatment groups at 21-day in terva l s during the experiment. S tra in D i e t 1 Time on diet (days) 0 D 21 D 42 D 63 D Quesnel LC 1 34.3 1 54.1 178.0 201.1 ± 2.2 ± 0.9 ± 2.4 ± 4.4 Quesnel HC 135.3 1 54.6 170.9 193.7 ± 0.9 ± 5.7 ± 0.3 ± 7.4 B. Qual . LC 108.3 122.7 1 33.8 1 46.5 ± 1.8 ± 1 . 2 ± 2.3 ± 6.0 B. Qual . HC 1 06. 1 112.6 118.0 1 35.4 ± 4.3 ± 8 . 1 ± 7.8 ± 9.9 Rob.Crk. LC 115.7 127.3 143.6 165.6 ± 3.3 ± 3.8 ± 5.7 ± 9.2 Rob.Crk. HC 116.1 124.2 1 32.8 151.6 ± 3.8 ± 2.7 ± 2.0 ± 6.7 Means ± SEM (standard error of the mean) 1 Diet LC = low carbohydrate (control) d i e t ; Diet HC = high carbohydrate d i e t . There were no s i g n i f i c a n t ef fects of diet on mean bodyweights of chinook salmon within the Quesnel, Big Qualicum or Robertson Creek s t r a i n s , at days 21, 42 or 63 of the growth t r i a l . 5 6 In the individual analyses of variance, by s t r a i n , there were no s i g n i f i c a n t differences on day 21, day 42 or day 63 for mean body weights between the high carbohydrate and control groups within any of the three chinook strains tested. Nevertheless, chinook of each s t r a i n exhibited a general trend of reduced body weight in groups fed the high carbohydrate d i e t . Average body weight gains (Table 6) revealed the same trend, however, differences were not s i g n i f i c a n t due to va r i a t i o n between replicate groups. Sp e c i f i c growth rates (SGR, [(LnW2 - LnW1) * T] x 100) over the 63 day feeding period are shown in Table 7 and Figure 4. Specific growth rates were calculated for each 21 day i n t e r v a l of the feeding t r i a l , between weighings (SGR-1 for days 0 -21, SGR-2 for days 21-42, and SGR-3 for days 42-63) and for the entire experimental feeding period (SGR-63D). Individual analyses (by strain) of s p e c i f i c growth rates (SGR-1, SGR-2 and SGR-3), revealed s i g n i f i c a n t reductions in the second in t e r v a l s p e c i f i c growth rate (SGR-2) of high carbohydrate-fed f i s h , in both Big Qualicum and Robertson Creek strains (p<0.05). A similar but non-s i g n i f i c a n t trend was also seen in the SGR-1 of these two stra i n s , and in the SGR-2 of Quesnel chinook fed the high carbohydrate d i e t . S p e c i f i c growth rates (SGR-3) of high carbohydrate-fed f i s h during the t h i r d i n t e r v a l were comparable to those of control-fed f i s h , in each of the chinook s t r a i n s . Quesnel chinook exhibited a less 57 Table 6 - Average body weight gains (BWG), and percent BWG of treatment groups at the end of the 63-day feeding per iod . S tra in D i e t 1 BWG(g) 2 %BWG Quesnel LC 66.6 + 2.2 49.7 Quesnel HC 58.9 + 8.2 43.2 B .Qual . LC . 38.2 + 4.2 35.3 B .Qual . HC 29.5 + 5.7 27.6 Rob.Cr. LC 50. 1 + 6.0 43. 1 Rob.Cr. HC 35.4 + 2.8 30.6 Means ± SEM (standard error of the mean) Diet LC = low carbohydrate (control) d i e t ; Diet HC = high carbohydrate d i e t . There were no s i g n i f i c a n t e f fects of d iet on average body weight gains of chinook salmon within the Quesnel} Big Qualicum or Robertson Creek s t r a i n s . 58 Table 7 - Spec i f i c Growth Rates of treatment groups over 21-day in terva l s and the 63-day duration of the feeding t r i a l . S tra in D i e t 1 2SGR-1 2SGR-2 2SGR-3 2SGR-63D Quesnel LC .725 .759 .636 .707 ± . 0 8 0 ± . 0 7 0 + .031 ± . 0 0 7 Quesnel HC .692 .540 .669 .634 ± . 1 1 3 ± . 1 32 + .146 ± . 0 5 5 B. Qual . LC .660 .457 .470 .529 ± . 0 2 7 ± . 0 2 9 ± . 0 8 9 ± . 0 3 0 B. Qual . HC .31 1 * * .235 .734 .426 ± . 1 1 5 ± . 0 2 1 ± . 0 2 9 ± . 0 4 1 Rob.Crk. LC .505 .634 .748 .629 ± . 0 0 7 ± . 0 3 5 ± . 0 6 0 ± . 0 3 4 Rob.Crk. HC .354 * * .353 .689 .466 ± . 0 4 2 ± . 0 2 5 ± - 1 0 6 ± . 0 1 3 Means ± SEM (standard error of the mean) 1 Diet LC = low carbohydrate (control) d i e t ; Diet HC = high carbohydrate d i e t . 2 Spec i f i c Growth Rate = [ (lnW2 - lnW1) + T ] x 100 where W2 = f i n a l weight (g), W1 = i n i t i a l weight (g), and T = time in days. SGR-1 = SGR for days 0 - 2 1 ; SGR-2 = SGR for days 21 - 42; SGR-3 = SGR for days 42 - 63; and SGR-63D = SGR for days 0 - 6 3 . **Signi f i cant at the a=0.05 l e v e l . 59 Figure 4 : Twenty-one Day Specific Growth Rates in Chinook Salmon of Three Strains Fed Low VS High Carbohydrate Diets. 1.000 o 0.800 a: *$ 0.600-i 2 o 0 0.400 H u= "o S. 0.200-CO LOW CARB. DIET • HIGH CARB. DIET LZ2 0.000 1.000 QUESNEL means ± 1 s.e.m 1 1 SGR-1 SGR-2 SGR-3 3 o 0.800-j •5 0.600 8 o o V: *o § . 0.200 to 0.400 0.000 1.000 BIG QUAUCUM 1 means ± 1 s.e.m.1 £1 SGR-1 SGR-2 SGR-3 o 0.800-x: % 0.600 8 0 0 0.400 'o S. 0.200 CO 0.000 ROBERTSON CREEK means ± 1 s.e.m.1 1 1 1 (A (A SGR-1 SGR-2 SGR-3 Day 0-21 Day 21-42 Day 42-63 1 Means ± 1 standard error of the mean. N - 2 . •Significant at p < 0.05 60 pronounced deviat ion in spec i f i c growth rates between d ie t groups, and no s i g n i f i c a n t di f ferences in SGR were found. O v e r a l l spec i f i c growth rates (SGR-63D) were not s i g n i f i c a n t l y influenced by d i e t . Condit ion factor (Table 8), defined as body weight x 100 -s- (fork l e n g t h ) 3 , was not s i g n i f i c a n t l y influenced by d ie t in the ind iv idua l s t r a i n analyses. 4.2.3 Carcass composition I n i t i a l and f i n a l (day 63) mean carcass compositions (percent dry matter, crude p r o t e i n , crude fat and ash) are given in Table 9. Carcass composition data (% dry matter basis) exhibi ted a consistent trend of increased body prote in and ash, and decreased body fa t , in high carbohydrate-fed f i sh r e l a t i v e to contro l - f ed f i s h , in each of the three chinook s tra ins (see Figure 5) . Percent body water a lso exhibited a s l i g h t upward tendency in high carbohydrate d iet groups. These ' trends' were s t a t i s t i c a l l y s i g n i f i c a n t between d ie t groups in only the fol lowing analyses: f i n a l % carcass prote in in Big Qualicum chinook and Robertson Creek chinook (p<0.0l); and f i n a l % carcass fat in Robertson Creek chinook (p<0.05). A l l other comparisons revealed no s i g n i f i c a n t di f ferences (p>0.05). 61 Table 8 - Mean condit ion factors in treatment groups, i n i t i a l l y and at the end of the 63-day feeding per iod . S tra in - D i e t 1 Od-Cond.F. -* 63d-Cond.F. Quesnel - LC 1 .37 ± . 0 1 1 .45 ± . 0 0 Quesnel - HC 1 .40 ± . 0 3 1 .45 ± . 0 6 B .Qual . - LC 1 .25 ± . 0 2 1.31 ± . 0 3 B .Qual . - HC 1.23 ± . 0 3 1 .30 ± . 0 4 Rob.Cr. - LC 1 .29 ± . 0 3 1 .37 ± . 0 5 Rob.Cr. - HC 1 .30 ± . 0 1 1 .34 ± . 0 4 Means ± SEM (standard error of the mean) Diet LC = low carbohydrate (control) d i e t ; Diet HC = high carbohydrate d i e t . Condit ion factor = body weight x 100 T (fork l e n g t h ) 3 . There were no s i g n i f i c a n t ef fects of d iet on f i n a l (day 63) condit ion factors of chinook salmon within the Quesnel, Big Qualicum or Robertson Creek s t r a i n s . 62 Table 9 - Proximate analyses of whole f i s h S-D 2 %Dry Matter %Protein 3 %Lipid 3 %Ash3 Od 4 63d Od 63d Od 63d Od 63d Qs-LC \ 27.36 58.35 27.70 7.68 24.84 ± . 5 1 61 .96 ±1 .91 23.03 ±1 .45 8.86 ± . 1 0 ± . 2 7 ± . 4 5 ± . 6 6 ± . 2 8 Qs-HC / 26.55 60.84 23.07 8.83 ± 2 . 0 3 ± 2 . 5 9 ± 1 . 0 1 ± . 5 0 BQ-LC \ 25.79 63.00 21 .77 8.45 22.67 ± . 2 9 68.06 ± . 1 0 15.49 ± . 9 9 9.95 ± . 1 7 ± . 3 4 ± . 6 1 , .^ ± . 9 4 ± . 2 5 BQ-HC / 24.72 65.58 K -X 19.78 9.43 ± . 6 2 ± . 0 8 ±1 .03 ± . 1 9 RC-LC \ 27.07 58.72 24.86 7.77 24.24 ± . 0 6 64.45 ± . 4 8 18.86 ± . 7 5 9.05 ± . 2 1 ± . 4 5 ±1 .23 ± 1 - 7 3 18.74** ± . 2 9 RC-HC / 25.03 65.97 *** 9.40 ± . 8 0 ± . 0 5 ±1 .00 ± . 6 3 Means ± SEM (standard error of the mean) 1 Analys i s of 8 whole f i s h from each chinook s t r a i n at day 0 (2 f i s h per tank), and 8 whole f i s h from each s t r a i n -diet group at day 63 (4 f i s h per rep l i ca te tank) . Note: Pr ior to day 0, a l l f i s h had been fed only contro l Diet 1. Therefore resu l t s from the 4 tanks for each s t r a i n were averaged. F i s h sampled within any given tank were pooled p r i o r to a n a l y s i s . 2 S-D = Chinook s t r a i n and diet combination: Qs = Quesnel; BQ = Big Qualicum; RC = Robertson Creek; Diet LC = low carbohydrate (control) d i e t ; Diet HC = high carbohydrate d i e t . 3 Dry matter bas i s . 4 Time on diet (days'). **Signi f i cant at the a=0.05 l e v e l . ***Signi f icant at the a=0.0l l e v e l . Figure 5: Effect of Dietary Carbohydrate on Body Composition of Chinook Salmon of Three Strains Fed Low or High Carbohydrate Diets for 63 Days. 80-a & c 3 £ Q. a a o a o 70 60 50 40 Initial 1=1 Day 63 - Low Carta. Diet LTD Day 63 - High Carta. Diet C33 ** means ± 1 s.e.m.1 a a OJ o a u LC HC LC HC LC HC QUESNEL BIG QUALICUM ROBERTSON CR. 1 Means ± 1 standard error of the mean, N » 2 . * Significant at p < 0.05. * * Significant at p < 0.01. 64 4.2.3.1 Growth in Terms of Prote in and L i p i d Instantaneous prote in gains (IPG, % gain in prote in /day , as a percentage of t o t a l body protein) and instantaneous l i p i d gains (ILG, % gain in l i p i d / d a y , as a percentage of t o t a l body l i p i d ) for the treatment groups over the 63 day feeding t r i a l are shown in Table 10. IPG and ILG were ca lcu la ted to make comparisons of t i ssue deposit ion rates between treatment groups. In general , f i sh fed the high carbohydrate diet demonstrated a lower l i p i d deposi t ion rate than those fed the contro l d i e t , however, th i s d i f ference was only s i g n i f i c a n t in the analys i s of Robertson Creek chinook (p<0.05). There were no s i g n i f i c a n t e f fects of d ie t on IPG. F i n a l (day 63) chinook body weights were a lso compared on the basis of average weights of protein only (Table 11 and Figure 6). Indiv idual s t r a i n analyses revealed no s i g n i f i c a n t ef fects of d iet on prote in weights. 4.2.4 Feed Intake and E f f i c i e n c y Indices The average feed intake per f i s h (FI) , feed e f f i c i ency (FE, g wet body weight gain -s- g feed intake) , and energy e f f i c i e n c y (EE, carcass gross energy gain + feed gross energy intake) ra t io s of the treatment groups over the 63 day feeding period are provided in Table 12. FE and EE ra t io s are also i l l u s t r a t e d in Figure 7. Although there appears to be a trend for decreased feed intake in groups fed the high carbohydrate d iet r e l a t i v e to 65 Table 10 - Instantaneous prote in gain (IPG) and instantaneous l i p i d gain (ILG) in treatment groups over the 63-day feeding per iod . S tra in D i e t 1 IPG^ ILG Quesnel LC .696 ± . 0 0 9 1 .084 ± . 0 8 6 Quesnel HC .644 ± . 0 1 2 .675 ± . 0 8 5 B .Qual . LC .560 ± . 0 1 0 1 .221 ± . 0 9 1 B .Qual . HC .464 ± . 0 1 0 .908 ± . 0 4 9 Rob.Cr. LC .597 ± . 0 3 8 1 .183 ± . 0 0 6 Rob.Cr. HC .535 ± . 0 4 2 .486 * * ± . 1 0 3 Means ± SEM (standard error of the mean) 1 Diet LC = low carbohydrate (control) d i e t ; Diet HC = high carbohydrate d i e t . 2 Instantaneous prote in gain = [ (lnP2 - l n P ^ + T ] x 100 where P2=final weight of p r o t e i n , P ^ i n i t i a l weight of p r o t e i n , and T=time in days. There were no s i g n i f i c a n t e f fec ts of d ie t on IPG in chinook salmon within the Quesnel, Big Qualicum or Robertson Creek s t r a i n s . 3 Instantaneous l i p i d gain = [ ( l n L 2 - l n L 1 ) * T ] x 100 where L2=final weight of l i p i d , L i = i n i t i a l weight of l i p i d , and T=time in days. **Signi f i cant at the a=0.05 l e v e l . 6 6 Table 11 - Mean prote in weights of chinook in treatment groups i n i t i a l l y and at the end of the 63 day feeding t r i a l . S -Die t 1 Body Protein Weights Od 63d Qs - LC 20.7 ± . 2 3 32.0 ± . 1 7 Qs - HC 20.8 ± . 1 0 31.2 ± . 0 9 BQ - LC 16.7 ± . 2 0 23.8 ± . 8 5 BQ - HC 16.4 ± . 4 7 21 .9 + .77 RC - LC 18.1 ± . 3 6 26.3 ± 1.15 RC - HC 18.1 ± . 4 2 25.0 ± . 2 1 Means ± SEM (standard error of the mean) S-Diet = chinook s t r a i n and diet combination: Qs = Quesnel; BQ = Big Qualicum; and RC = Robertson Creek. Diet 1 = LC (low carbohydrate) contro l d i e t ; Diet 2 = HC (high carbohydrate) experimental d i e t . Average weights of body prote in only . There were no s i g n i f i c a n t ef fects of d iet on f i n a l (day 63) prote in weights in chinook salmon within the Quesnel, Big Qualicum or Robertson Creek s t r a i n s . 67 Table 12 - Feed intake (FI ) , feed e f f i c i ency (FE) and energy e f f i c i e n c y (EE) ra t io s for treatment groups at the end of the 63-day feeding per iod . S -Die t 1 F I ( g ) 2 F E 3 E E 4 Qs - LC 111.50 ± 2.02 .597 ± . 0 0 6 .258 ± . 0 0 6 Qs - HC 107.63 ± 1 3 . 8 3 .547 * * ± . 0 0 5 .218 ± . 0 3 7 BQ - LC 70.08 ± 7.52 .545 ± . 0 0 1 .230 ± . 0 0 1 BQ - HC 62.46 ± 9.86 .469 * * ± . 0 1 2 .234 ± . 0 1 6 RC - LC 86.55 ± 5.85 .576 ± . 0 2 1 .256 ± . 0 1 9 RC - HC 71 .43 ± 1 .34 • .530 ± . 0 0 5 .181 ± . 0 2 5 Means ± SEM (standard error of the mean) 1 S-Diet = chinook s t r a i n and diet combination: Qs = Quesnel; BQ = Big Qualicum; and RC = Robertson Creek. Diet LC = low carbohydrate (control) d i e t ; Diet HC = high carbohydrate d i e t . 2 Feed intake = average intake per f i s h (g) over 63 days. 3 Feed e f f i c i e n c y = wet body weight gain (g) -J- feed intake (g). 4 Energy e f f i c i e n c y = carcass gross energy gain (kcal) feed GE intake (kca l ) . Gross energy values used for c a l c u l a t i o n s were: p r o t e i n , 5.65 k c a l / g ; l i p i d , 9.5 k c a l / g ; ge la t in i zed wheat starch and a - c e l l u l o s e , 4.20 k c a l / g ; and ni trogen-free ex trac t , 3.90 kca l /g (Lloyd et al., 1978). There were no s i g n i f i c a n t e f fects of diet on feed intakes or energy e f f i c i e n c y ra t ios in chinook salmon within the Quesnel, Big Qualicum or Robertson Creek s t r a i n s . **Signi f i cant at the a=0.05 l e v e l . Figure 6 : Protein Weights of Chinook Salmon Fed Low or High Carbohydrate Diets for 63 Days. •Z) Low Carbohydrate Diet — Day 63 means ± 1 s.e.m. IZZ High Carbohydrate Diet - Day 63 69 Figure 7: Feed Efficiency and Energy Efficiency Ratios of Chinook Salmon of Three Strains Fed Low or High Carbohydrate Diets for 63 Days. 0.750 11 •o u c £ o 1 = 0.500 • 0.250 Diets: QD Low Carbohydrate E23 High Cabohydrate 21 Quesnel B. Qualicum means ± 1 s.e.m. Robertson Cr. 0.300 ^ ^ 0.250 § u § ^ 0.200 o cn | & 0.150 0.100 Quesnel B. Qualicum Robertson Cr. 1 Means ± 1 standard error of the mean, N=2. "'Significant at p < 0.05. 70 c o n t r o l s , d ie t d id not s i g n i f i c a n t l y affect feed consumption in the i n d i v i d u a l s t r a i n analyses . Feed e f f i c i e n c i e s were general ly reduced in f i sh fed the high carbohydrate d i e t . Indiv idual analyses revealed s i g n i f i c a n t reductions in feed e f f i c i ency in Quesnel and Big Qualicum chinook fed the high carbohydrate d ie t r e l a t i v e to respect ive contro l groups (p<0.05). Mean energy e f f i c i e n c y ra t io s were lower for f i sh fed the high carbohydrate d ie t r e l a t i v e to those of contro l groups in the Quesnel and Robertson Creek chinook. However, i n d i v i d u a l s t r a i n analyses showed no s i g n i f i c a n t af fects of d ie t on EE, due to high v a r i a t i o n among r e p l i c a t e groups. 4.2.5 Prote in U t i l i z a t i o n Prote in e f f i c i ency r a t i o s {PER, g body weight gain -s- g prote in intake) and productive prote in values (PPV, g prote in gain -r g prote in intake) are provided in Table 13 and Figure 8. In the i n d i v i d u a l s t r a i n analyses, PER values were s i g n i f i c a n t l y reduced in Quesnel and Big Qualicum chinook fed the high carbohydrate d ie t r e l a t i v e to respect ive contro ls (p<0.05), ( in accordance with the e f fects of d iet on feed e f f i c i e n c y in these s t r a i n s ) . Robertson Creek chinook diet groups did not d i f f e r s i g n i f i c a n t l y with respect to PER. Productive prote in values were not s i g n i f i c a n t l y influenced by d ie t in any of the three chinook s t r a i n s . 71 Table 13 - Protein e f f i c i ency ra t io s (PER) and productive prote in values (PPV) in treatment groups over the 63-day feeding per iod . S tra in D i e t 1 PER^ PPV Quesnel LC 1 .39 ± . 0 2 .261 ± . 0 1 0 Quesnel HC 1 .27 * * ± . 0 2 .246 ± . 0 3 0 B.Qual . LC 1 .27 ± . 0 0 .252 ± . 0 0 2 B .Qual . HC 1 .09 ± . 0 4 .233 ± . 0 1 0 Rob.Cr. LC 1 .34 ± . 0 7 .252 ± . 0 1 2 Rob.Cr. HC 1.15 ± . 0 7 .246 ± . 0 1 2 Means ± SEM (standard error of the mean) 1 Diet LC = low carbohydrate (control) d i e t ; Diet HC = high carbohydrate d i e t . 2 Prote in e f f i c i ency r a t i o = body weight gain (g) * prote in intake (g). 3 Productive prote in value = prote in gain (g) + prote in intake (g). There were no s i g n i f i c a n t e f fects of d iet on PPV in chinook salmon of the Quesnel, Big Qualicum or Robertson Creek s t r a i n s . **Signi f i cant at the a=0.05 l e v e l . 72 Figure 8 : Protein Efficiency Ratios and Productive Protein Values of Chinook Salmon of Three Strains Fed Low or High Carbohydrate Diets for 63 Days. 2.00 % I 1.50 + I g 1.00-• 1 * 0.50 + 0.00 Diets: CZ3 Low Carbohydrate 7Z2 High Cabohydrate V? 'A Quesnel B. Qualicum means ± 1 s.e.m. Robertson Cr. 0.40 _ 0.35 | | 0.30 + f | 0.25 + % 8 £ £ 0.20 + | §> 0.15 + e I 0.10+ °- i 0.05 + 0.00 0 2 Quesnel P7 id B. Qualicum means ± 1 s.e.m Robertson Cr. 1 Means ± 1 standard error of the mean. N=2. •Significant at p < 0.05. 73 4.2.6 L iver Glycogen and Hepatosomatic Index Percent l i v e r glycogen leve l s (LG) and hepatosomatic indices (HSI, l i v e r weight:body weight ra t ios ) are given in Table 14 for day 0, day 63 (end of experimental feeding period) and day 84 (end of 21 day feed withdrawal per iod) . A d d i t i o n a l l y , day 63 HSI were ca lcu la ted with the exclusion of l i v e r glycogen (HSI-G), to examine the ef fect of the treatments on l i v e r weight independent of a l t e r a t i o n s in l i v e r glycogen l e v e l . L iver glycogen leve l s are presented graph ica l ly in Figure 9. Arcsine transformation was c a r r i e d out on these data p r i o r to analyses of var iance . Indiv idual s t r a i n analyses revealed higher HSI in chinook of the Quesnel and Robertson Creek s t ra ins fed high carbohydrate d iet (p<0.01), than in respect ive contro l groups. Big Qualicum chinook exhibi ted no d ie tary influences on HSI. Analyzing HSI with l i v e r glycogen excluded (HSI-G) e s s e n t i a l l y d id not change the r e s u l t s , but only the s ign i f i cance p r o b a b i l i t y l eve l of a (a=0.05). At 21 days post feed withdrawal (day 84 of experiment), a l l HSI had decl ined to (or below) i n i t i a l values and no s i g n i f i c a n t d i f ferences were found between d ie tary treatment groups. L iver glycogen l eve l s were s i g n i f i c a n t l y elevated in the groups fed high carbohydrate d iet in the Quesnel and Robertson Creek chinook (p<0.05) in the ind iv idua l analyses. Percent l i v e r glycogen l eve l s ranged from 3.2 to 11.3 in Quesnel chinook, from 2 to 7.5 in Big Qualicum chinook, and 74 Table 14 - Mean percent l i v e r glycogen (LG) l eve l s and hepatosomatic indices (HSI) in treatment groups ( i n i t i a l l y , during feeding and 21 days post feed withdrawal). S-D 1 % L iver Glycogen Hepatosomatic Index^ HSI-G Od 63d 84d 4 Od 63d 84d 4 63d Qs-LC 1 .34 ± . 5 3 2.18 ± . 2 2 0.73 ± . 0 9 1 .36 ± . 0 5 1 .48 ± . 0 5 1 .24 ± . 1 2 1 .46 ± . 0 5 Qs-HC 1 .51 ± . 2 6 * * * 7.32 ± . 9 5 0.84 ± . 4 5 1 .33 ± . 0 6 2.08** ± . 0 4 1 .26 ± . 0 9 .91** ± . 0 5 BQ-LC 1 .64 ± . 4 7 1.13 ± . 0 9 0.41 ± . 1 2 1 .59 ± . 1 0 1 .98 ± . 0 5 1 .29 ± . 0 3 1 .94 ± . 0 3 BQ-HC 1 .36 ± . 2 1 3.59 ±1 .06 0.24 ± . 0 3 1 .67 ± . 0 1 1 .95 ± . 0 8 1 .30 ± . 0 2 1 .86 ± . 0 5 RC-LC 1 .42 ± . 2 3 1 .40 ± . 0 1 0.48 ± . 0 7 1 .37 ± . 0 3 1 .62 ± . 0 1 1 .35 ± . 0 9 1 .59 ± . 0 1 RC-HC 1 .62 ± . 0 2 5.25*** ± . 4 8 0.39 ± . 0 2 1 .44 ± . 0 6 1.94** ± . 0 2 1 .43 ± . 1 2 .85** ± . 0 3 Means ± SEM (standard error of the mean) 1 S-D = Chinook s t r a i n and diet combination: Qs = Quesnel; BQ = Big Qualicum; and RC = Robertson Creek Diet LC = low carbohydrate (control) d i e t ; Diet HC = high carbohydrate d i e t . 2 HSI = L iver weight:body weight r a t i o . 3 HSI-G = HSI reca lculated with l i v e r glycogen weight excluded. 4 Day 84 was the end of a 21 day feed withdrawal period (from day 63 to day 84). There were no s i g n i f i c a n t di f ferences between d ie t groups in HSI or %LG l eve l s in chinook salmon 21 days after feed withdrawal within the Quesnel, Big Qualicum or Robertson Creek s t r a i n s . **Signi f i cant at the a=0.05 l e v e l . ***Signi f icant at the a=0.0l l e v e l . Figure 9 10 c Q> O O o > 9-8-7-6-5-4-3-2-1 -0 Effect of Dietary Carbohydrate on Liver Glycogen Level in Chinook Salmon of Three Strains: During Feeding and subsequent to feed withdrawal. STRAIN: Low Carb. Quesnel LZ3 B. Qualicum LZZ1 Robertson Cr \ZS High Carb. rxzi 63 Time (days) ^ Means ± 1 standard error of the mean, N—2. *Significant at p < 0.01. means ± 1 s.e.m 1 84 ( 21 days after feed withdrawal ) 76 from 2.2 to 8.9 in Robertson Creek chinook fed the high carbohydrate d i e t . After 21 days of feed withdrawal, a l l LG decreased to basal l e v e l s , with no s i g n i f i c a n t di f ferences between treatments groups (in the o v e r a l l or i n d i v i d u a l analyses of var iance) . 4.2.9 M o r t a l i t y M o r t a l i t y in chinook of the Quesnel and Robertson Creek s t ra ins was neg l i g ib l e in a l l groups. Big Qualicum chinook experienced morta l i ty p r i m a r i l y in the l a t t e r part of the growth t r i a l (Table 15), with a t o t a l of 6 m o r t a l i t i e s in contro l groups (3 per r e p l i c a t e ) , and 11 m o r t a l i t i e s in groups fed the high carbohydrate d iet (6 in one r e p l i c a t e , and 5 in the o ther) . 77 Table 15 - Tota l m o r t a l i t i e s in treatment groups over the 63-day feeding p e r i o d . S tra in D i e t 1 Tota l M o r t a l i t i e s (NO. Of f i sh) Quesnel LC 1 ( 53 ) Quesnel HC 0 ( 52 ) Big Qualicum LC 6 ( 60 ) Big Qualicum HC 1 1 ( 60 ) Robertson C r . LC 1 ( 60 ) Robertson C r . HC 2 ( 60 ) Diet LC = low carbohydrate (control) d i e t ; Diet HC = high carbohydrate d i e t . 78 4.3 DISCUSSION 4.3.1 General In th i s study, chinook salmon of the d i f f erent s tra ins examined, d i f f e r e d s i g n i f i c a n t l y in mean body weights, and degrees of v a r i a t i o n around the means. Genetic di f ferences in growth p o t e n t i a l , time of s m o l t i f i c a t i o n and behaviour (Withler et al., 1987; Clarke and Shelbourn, 1985; Taylor and L a r k i n , 1986), as well as the extended holding period and r e s t r i c t e d feeding contributed to these s ize d i f f erences . On the other hand, i n i t i a l body weights of the four experimental groups within each chinook s t r a i n , d id not d i f f e r s i g n i f i c a n t l y . As d i f ferences in s ize were associated with d i f f erent chinook s t r a i n s , the two var iab les were confounded, and s ize could not be adjusted or corrected for in the experimental analyses. Therefore chinook of each s t r a i n were assessed i n d i v i d u a l l y for carbohydrate u t i l i z a t i o n . Any attempted comparison between chinook of the d i f f erent s tra ins in th i s study must be made in l i g h t of the fact that body weight i s corre la ted with metabolism. Data from Shuswap chinook were not included in analyses due to serious b a c t e r i a l kidney disease outbreak in a l l experimental groups of th i s s t r a i n . 79 4.3.2 Growth Rates Although the trends in body weight suggest a reduction in growth in chinook fed the high carbohydrate d i e t , d i f ferences were not s i g n i f i c a n t within Quesnel, Big Qualicum or Robertson Creek chinook s t r a i n s . This f inding was confirmed in the examination of body weight gains over the 63 day feeding t r i a l , which were not s i g n i f i c a n t l y a l t ered by diet in the three s t r a i n s . More revea l ing , however, are the analyses of spec i f i c growth rates (SGR, percent gain in body weight per day) during the three 21-day in terva l s between weighings. Figure 4 c l e a r l y i l l u s t r a t e s a dec l ine , p a r t i c u l a r l y in the second growth i n t e r v a l , in s p e c i f i c growth rates of high carbohydrate-fed chinook r e l a t i v e to those of respect ive contro l groups. Following th i s dec l ine , SGR improved in the t h i r d growth i n t e r v a l , and were comparable to those of contro l groups. Figure 3 a lso depicts th i s trend, as growth curves diverge in the f i r s t and second growth i n t e r v a l s , then exhib i t s imi lar slopes in the t h i r d growth i n t e r v a l . This indicates that an adaptation response to the high carbohydrate diet occurred within the 63 day duration of the growth t r i a l . The narrower margin of d i f ference in f i n a l mean weights between groups fed the high and low carbohydrate d iets in Quesnel chinook suggests a s t r a i n di f ference in response to the high carbohydrate d i e t . 80 4.3.3 Carcass Composition Figure 5 i l l u s t r a t e s the changes in carcass composition of chinook over the 63-day feeding t r i a l . I n i t i a l carcass composition data are shown for comparative purposes. F i sh fed the high carbohydrate d ie t general ly had lower body f a t , but higher body prote in and ash than those fed contro l d i e t . S imi lar responses to high carbohydrate d iets have a lso been demonstrated in studies on rainbow trout (Beamish et al., 1986; H i l t o n and Atkinson, 1982; Refs t ie and Austreng, 1981). The d ie tary ef fects were s i g n i f i c a n t in Big Qualicum chinook for % carcass p r o t e i n , and in Robertson Creek chinook for both % carcass prote in and % carcass f a t . Chinook fed the contro l d ie t a lso had s l i g h t l y higher but non-s ign i f i cant % carcass dry matter l eve l s than noted for the high carbohydrate-fed groups. 4.3.3.1 Growth in Terms of Carcass Composition In view of the dif ferences in carcass composition, body weights were re-examined on the basis of prote in weight, as shown in Figure 6. F i n a l prote in weights were not s i g n i f i c a n t l y affected by d i e t , ind ica t ing that a s imi lar amount of ( tota l ) prote in was l a i d down when chinook were fed to s a t i a t i o n on e i ther the low or high carbohydrate d i e t s . Instantaneous prote in and l i p i d gains (Table 10) were ca lcu la ted to describe the d a i l y rates of prote in and fat deposit ion (percent fat gain per day and percent prote in 81 gain per day, respect ive ly) r e l a t i v e to i n i t i a l l e v e l s . These indices were ca lcu la ted in the same manner as spec i f i c growth rate , using the natural logarithms of i n i t i a l and f i n a l prote in weights or fat weights. Instantaneous prote in gains were cons i s tent ly lower in f i sh fed the high carbohydrate diet but d ie tary di f ferences were not s i g n i f i c a n t in any one of the three chinook s t ra ins tested, as confirmed by the f i n a l prote in weights. In the i n d i v i d u a l s t r a i n analyses only Robertson Creek chinook exhibi ted s i g n i f i c a n t l y lower IFG in groups fed the high carbohydrate d i e t . Big Qualicum chinook, on the other hand, d isplayed the greatest r e l a t i v e fat gains o v e r a l l , and the narrowest margin of d i f ference between d ie t groups. 4.3.4 Feed Intake and Feeding Response Feed intake was not s i g n i f i c a n t l y affected by d i e t , although feed consumption was general ly lower for f i sh fed the high carbohydrate d i e t . This p a r t i a l l y explains the o v e r a l l reduction in growth on the high carbohydrate d i e t . In Robertson Creek chinook, the margin of d i f ference in feed intakes between diet groups was greater than in the other two s t r a i n s , and both r e p l i c a t e groups fed the high carbohydrate diet exhibi ted cons i s tent ly lower feed intakes than contro l groups. Nonetheless, high v a r i a b i l i t y between r e p l i c a t e groups obscured any po tent ia l s i g n i f i c a n t d i f f erences . Feed intake has been shown to decl ine in 82 rainbow trout fed high l eve l s of d i g e s t i b l e carbohydrate in the form of extruded starch (Hil ton and S l i n g e r , 1983). Feeding responses of f i s h fed the high carbohydrate d iet d i f f e r e d noticeably from those of the contro l groups, during the course of the feeding t r i a l . Observations included a reduced enthusiasm for the high carbohydrate feed accompanied by increased s p i t t i n g of feed p e l l e t s as s a t i a t i o n was approached. It was consequently more d i f f i c u l t to determine when s a t i a t i o n was reached in these groups. In teres t ing ly , chinook from d i f f erent s t ra ins d id not behave equally in th i s regard. While Robertson Creek chinook exhibi ted the most conspicuous di f ferences in feeding response between diet groups, Quesnel chinook diet groups were d i f f i c u l t to d i s t i n g u i s h . In fac t , the feed intake of one of the high carbohydrate rep l i ca te s of Quesnel chinook ac tua l ly exceeded that of the contro l groups. In addi t ion to d ie tary induced d i f ferences , chinook s tra ins could be d is t inguished in terms of general feeding behaviour and e x c i t a b i l i t y . Thus accurate feeding of a l l groups to sa t ia t ion proved d i f f i c u l t . 4.3.5 Feed and Energy E f f i c i e n c y The observed dif ferences in feed e f f i c i e n c i e s (wet body weight gain -f feed intake) among the d i f f erent chinook s tra ins (Figure 7) were l i k e l y associated with the s t r a i n di f ferences in body weight. F i s h s ize af fects metabolism and rate of growth. For example, smaller f i sh grow faster 83 on a percent body weight basis but require more energy per unit weight for metabolism than larger f i sh of the same species . Thus lower feed e f f i c i e n c y ra t io s would be expected in smaller f i s h due to the increased maintenance requirement r e l a t i v e to body weight (Halver, 1 9 8 9 ) . F i s h fed the high carbohydrate d iet had s i g n i f i c a n t l y lower feed e f f i c i ency ra t ios in both Quesnel and Big Qualicum chinook s t r a i n s . Energy e f f i c i ency means exhibited a downward trend in high-carbohydrate fed chinook of the Quesnel and Robertson Creek s t r a i n s , although data were highly v a r i a b l e , and the dietary di f ferences non-s ign i f i cant (Figure 7) . This trend indicates that a greater quantity of high carbohydrate d ie t was required to achieve ga in , and that less of the energy from th i s d iet was retained in the body. These reductions in feed and energy e f f i c i e n c y suggest poorer u t i l i z a t i o n of the carbohydrate. Feed and energy e f f i c i e n c i e s may have been underestimated due to feed loss , e spec ia l ly among high carbohydrate-fed groups. Health problems suffered by Big Qualicum chinook in the l a t t e r part of the growth t r i a l , l i k e l y affected feeding e f f i c i e n c y . In Quesnel and Robertson Creek chinook, on average, 26% of d ie tary gross energy was retained in the carcasses of c o n t r o l - f e d f i s h . This compares to an average of 22% for Quesnel chinook and 18% for Robertson Creek chinook in groups fed the high carbohydrate d i e t . Thus i t appears that f i s h retained less of the carbohydrate energy, whereas those 84 fed the contro l diet more readi ly converted excess energy to adipose t i s sue . The d ie t s employed in th i s study were formulated to be isonitrogenous and ( theore t i ca l l y ) isoenergetic (Table 2). The metabolizable energy values used to estimate ava i lab le energy of the two test d ie ts were; 4.5 kca l /g p r o t e i n , 8.5 kca l /g l i p i d , 3.8 kca l / g animal starch (Beamish et al., 1986), and 3.0 kca l /g g e l a t i n i z e d wheat s tarch (assuming a 75% d i g e s t i b i l i t y ) (Singh and Nose, 1967). The high carbohydrate d iet had 25% of i t s ME in the form of carbohydrate and 19% in the form of fat (30 % g e l a t i n i z e d wheat s tarch and 8% f i s h o i l , r e s p e c t i v e l y ) . By contras t , the c o n t r o l d ie t had 44% of i t s ME in the form of fat and a n e g l i g i b l e amount of ME (1.6%) from carbohydrate. However, H i l t o n et al . (1987), in the ir examination of carcass energy retent ion in rainbow t rout , reported overestimation of l i t e r a t u r e ME values for starch and glucose. In teres t ing , Big Qualicum chinook fed the high carbohydrate diet deposited more l i p i d r e l a t i v e to the i r i n i t i a l l i p i d l eve ls (see ILG, Table 10), narrowing the margin of di f ference in % carcass fat between d ie t groups, in comparison to the other s t r a i n s . This i s re f l ec ted in the higher average energy e f f i c i ency r a t i o of the high carbohydrate-fed groups (23%), which is s imi lar to the average value for contro l groups. Nevertheless, due to inconsis tencies among rep l i ca te groups, none of the 'apparent' d i f ferences were s t a t i s t i c a l l y s i g n i f i c a n t . 8 5 4.3.6 U t i l i z a t i o n of Protein Prote in e f f i c i ency ra t ios (wet weight gain * prote in intake) were s i g n i f i c a n t l y reduced in both Quesnel and Robertson Creek chinook fed the high carbohydrate d iet (Figure 8), consistent with the dif ferences in feed e f f i c i e n c i e s . However, PER values alone are of l i m i t e d usefulness in assessing prote in u t i l i z a t i o n , as they f a i l to take into account the nature of the weight ga in . Productive prote in values (protein gain prote in intake) , on the other hand, were not s i g n i f i c a n t l y af fected by diet (Figure 8) . This is confirmed by the analyses of f i n a l prote in weights and instantaneous prote in gains , which also revealed no s i g n i f i c a n t di f ferences between d ie t groups, within each s t r a i n , af ter 63 days on the test d i e t s . The s l i g h t but non-s igni f icant decl ine in f i n a l prote in weights and IPG of groups fed the high carbohydrate d ie t was probably due to the (non-s igni f icant) reduction in feed intake and hence growth, rather than a decl ine in prote in u t i l i z a t i o n . Importantly, these resul t s indicate that the high carbohydrate diet was as e f f ec t ive as the contro l d iet in sparing d ie tary prote in for prote in synthesis . Others have reported s imi lar f indings in rainbow trout (Kaushik and de Ol iva Te les , 1985; Pieper and P f e f f e r , 1980a, 1980b). 86 Greater f l u i d i t y in the feces of f i s h fed the high carbohydrate diet r e l a t i v e to contro l groups was observed during d i s sec t ion of f i s h , ind ica t ing an osmotic e f f ec t . 4.3.7 L iver Ef fec ts Percent l i v e r glycogen l eve l s (Figure 9) were elevated in a l l groups fed the high carbohydrate d i e t , but th i s was s i g n i f i c a n t only for Quesnel and Robertson Creek chinook (p<0.0l) . Quesnel chinook exhibi ted the highest l i v e r glycogen l eve l s among the three chinook s t ra ins tested, with i n d i v i d u a l values ranging from 3.2 to 11.3 %. This may be explained by the apparently better feed intake and growth rate on the high carbohydrate d iet in th i s s t r a i n . The greater e levat ion of hepatic glycogen d id not appear to have any detrimental effect on feeding, health or growth in Quesnel chinook over the nine week experimental per iod . The lower l i v e r glycogen l eve l s and higher r e l a t i v e fat deposit ion in Big Qualicum chinook fed high carbohydrate diet may indicate some dif ferences in metabolism in th i s s t r a i n . There were no obvious e f fects of d ie t on chinook l i v e r co lour . While HSI were elevated in chinook fed the high carbohydrate diet r e l a t i v e to contro l groups in both Quesnel and Robertson Creek s t r a i n s , Big Qualicum chinook exhibited e levat ion in both d ie t groups, and no s i g n i f i c a n t d i f ferences between diet groups. Refst ie and Austreng 87 (1981) found a s i g n i f i c a n t in terac t ion between trout family and d ie t for HSI. Analyses of HSI reca lculated with the weight of hepatic glycogen subtracted (HSI-G) had l i t t l e effect on the s t a t i s t i c a l outcome, changing only the a p r o b a b i l i t y l eve ls of s i g n i f i c a n t d i f f erences . Glycogen deposit ion was therefore only p a r t i a l l y responsible for increases in l i v e r weight. Feed withdrawal led to a decl ine in HSI to , or below, i n i t i a l values in each chinook s t r a i n . L iver glycogen l eve l s had decl ined to less than 1% in a l l groups 21 days after feed withdrawal. In rainbow trout s tudies , elevated l i v e r glycogen l eve l s and hepatosomatic indices in f i s h fed the high carbohydrate d i e t , dec l ined to normal values after 12 days of feed withdrawal (Hickl ing and March, 1982; H i l t o n , 1982). 4.3.8 M o r t a l i t y Health problems arose in two of the chinook s tra ins during the study. Shuswap chinook unfortunately had to be e l iminated from experimental analyses due to an outbreak of B a c t e r i a l Kidney Disease. Big Qualicum chinook suffered some morta l i ty in the l a t t e r t h i r d of the growth t r i a l , although most f i sh appeared vigorous during the study and those sampled had no obvious signs of disease. Higher morta l i ty was observed in Big Qualicum chinook fed the high carbohydrate d iet in comparison to contro l 88 groups. This may r e f l e c t a reduced a b i l i t y of high carbohydrate-fed f i sh to defend the body against i n f e c t i o n . High carbohydrate feeding, and hence elevated l i v e r glycogen, has been shown to cause sublethal e f fects in rainbow t rout , inc luding reduced resistence to copper (Dixon and H i l t o n , 1985) and selenium (Hi l ton and Hodson, 1983) t o x i c i t i e s . In growth t r i a l s , high d ie tary l eve l s of d i g e s t i b l e carbohydrate have been shown to increase ( P h i l l i p s et al., 1948) or have no effect on (Hi l ton and Atkinson, 1982; Austreng et al., 1977) morta l i ty in rainbow trout . In the present study, d iet exhibi ted no ef fect on morta l i ty in chinook of the Quesnel and Robertson Creek s t r a i n s , which remained healthy throughout the growth t r i a l . 8 9 4.4 CONCLUSIONS (Experiment 1) The resu l t s of th i s study indicate that chinook salmon are able to to lerate d i g e s t i b l e carbohydrate in the form of g e l a t i n i z e d wheat starch at a d ie tary l e v e l of 300 g/kg. Growth rates indicated an adaptation response to the carbohydrate within the 63 day experimental period in each of the Quesnel, Big Qualicum and Robertson Creek s t r a i n s . Consumption of a diet high in carbohydrate led to s i g n i f i c a n t changes in carcass composition, inc luding increased prote in and ash, and decreased fat r e l a t i v e to c o n t r o l s . Feed intake was s l i g h t l y but not s i g n i f i c a n t l y depressed in high carbohydrate groups and reduced appetite could be observed. Feed e f f i c i e n c y and energy e f f i c i e n c y exhibited a downward trend, although the l a t t e r was not s i g n i f i c a n t , due to high r e p l i c a t e v a r i a b i l i t y . It appears that less energy from carbohydrate was retained in the carcass than from fa t , as confirmed by the lower carcass fat depos i t ion . Importantly, prote in u t i l i z a t i o n was not adversely a f fec ted , i n d i c a t i n g a prote in sparing effect of the carbohydrate comparable to that of the l i p i d . Because the i n i t i a l body weights of chinook d i f f e r e d s i g n i f i c a n t l y between the s t ra ins tested, the resu l t s could not be ascribed purely to genetic di f ferences in carbohydrate u t i l i z a t i o n . Some observations however suggest inherent s t r a i n d i f f erences . For example, the superior growth rates of Quesnel chinook fed high carbohydrate 90 r e l a t i v e to contro l s , and better appetite for the high carbohydrate d i e t . L iver glycogen l eve l s and hepatosomatic indices were s i g n i f i c a n t l y elevated in high carbohydrate-fed f i s h of the Quesnel and Robertson Creek s t r a i n s . Big Qualicum chinook, however, showed no di f ferences in hepatosomatic indices between the two diets and had lower l i v e r glycogen concentrations in the high carbohydrate groups than chinook of the other s t r a i n s . This combined with the observation of higher r e l a t i v e body fat in these groups suggests a d i f ference in carbohydrate u t i l i z a t i o n by Big Qualicum chinook, compared with the other s t ra ins in the study. The higher energy e f f i c i ency displayed by high carbohydrate groups of th i s s t r a i n during the 63-day feeding period i s consistent with the lower l i v e r glycogen and higher body fat concentrat ions . Quesnel chinook, on the other hand, had the highest l i v e r glycogen concentrat ions , yet the best feeding and growth response on the high carbohydrate d i e t , ind ica t ing that during the time frame of the experiment, l i v e r glycogen concentrations of as high as 11.3% were to lerated by f i sh of th i s s t r a i n . The high carbohydrate had no ef fect on morta l i ty in Quesnel and Robertson Creek chinook. However, higher morta l i ty in Big Qualicum chinook fed the high carbohydrate d i e t , may indicate a reduced capacity to combat in fec t ion (assuming that the cause of morta l i ty was an i n f e c t i o n ) . 91 Longer term feeding t r i a l s , inc luding examination of key enzymes involved in carbohydrate metabolism for adaptation response, are recommended for future research. 92 5 EXPERIMENT 2 5.1 Part i ) Oral Glucose Tolerance in Selected Strains of B. C. Chinook Salmon. 5.1.1 MATERIALS AND METHODS 5.1.1.1 Tank Preparation and F i s h A l l o c a t i o n A ser ies of glucose tolerance t r i a l s was c a r r i e d out on 4 of the 7 remaining chinook stocks which had been held in the 2.5 m diameter 6000 1) outdoor tanks since March 16, 1988. The seawater flow rate was approximately 35 1/min per tank, DO ranged from 7.0 to 10.0 ppt, s a l i n i t y varied from 26 to 30 ppt and temperature decl ined from 10°C to 8 . 3 ° C over the course of the t r i a l s . Two to 3 weeks before t e s t ing a given stock, the f i s h were moved to a clean 8' diameter holding tank. The seawater supply was passed through a f ine nylon mesh to prevent any small edible organisms from entering the tank. In the glucose tolerance t e s t , blood i s sampled at various times after a glucose chal lenge. In order to minimize s tress , f i s h were held in separate tanks for the d i f f eren t sampling times (3, 6, 12, 18, 24, 30 and 36 hours after glucose admin i s tra t ion) . A sham group was also inc luded. Two banks of eight 200 1 f iberg lass tanks, each with a nylon mesh cover and a seawater flow rate of 5 1 /min, were set up in the indoor f a c i l i t y . The outer 2/3 of each tank top was covered with opaque material to minimize 93 disturbance. Water temperature was recorded throughout each t e s t . 5.1.1.3 Glucose Adminis trat ion: Prel iminary T r i a l To determine the best method of d e l i v e r i n g glucose to the f i s h , a prel iminary comparative t r i a l was conducted on October 26, 1988. Glucose was administered o r a l l y by intubation of e i ther a) ge la t in capsules f i l l e d with glucose powder or b) a concentrated glucose s o l u t i o n . Eighteen Robertson Creek chinook were d iv ided among six 200 1 indoor tanks, 3 f i s h per tank. The flow rate of seawater was 5 1/min/tank and water temperature was 10.1 °C. Treatment a) : One group of 3 f i s h was captured, s ing ly anesthetized in 2-phenoxyethanol (0.35 ml/1) then weighed (± 0.1 g ) . The glucose dose was adjusted on the basis of f i s h weight (167 mg glucose/100 g body weight). The dose was administered v ia 3 or 4 ge la t in capsules (s ize 2) p r e - f i l l e d with glucose monohydrate powder. The capsules were de l ivered through a 7 mm diameter p l a s t i c appl icator tube d i r e c t l y into the f i s h ' s stomach. F i sh weight, glucose dose and exact time of administrat ion were recorded. The f i s h were returned to the ir tank after recovery from anesthesia. This procedure was c a r r i e d out on the f i r s t 3 groups of f i s h . Treatment b): For the remaining 3 groups of f i s h , only the method of glucose de l ivery d i f f e r e d . Glucose was intubated d i r e c t l y into the f i s h ' s stomach through 2 mm diameter cannula tubing attached to a 100 ul glass syr inge . 94 The syringe was f i l l e d to appropriate volume with 80% glucose so lut ion (80 g glucose monohydrate/100 ml d i s t i l l e d water), ca lcu la ted according to the fol lowing equation: mis so lut ion = .167 g/100 g (glucose dosage rate) -f .8 g/ml (cone, of glucose solut ion) -f .909165 (cone, of glucose in glucose monohydrate powder) x body weight. Blood samples were taken at 2, 4 and 7 hours after glucose administrat ion from the 3 respective groups of f i s h in each treatment. Plasma glucose l eve l s were determined according to the protocol in sect ion 5.2 .6 . The resu l t s are shown in Figure 10. F i sh administered glucose in ge la t in capsules exhibi ted a slower esca lat ion of plasma glucose, r e l a t i v e to those given glucose in so lut ion form. This indicates that the time required for capsule breakdown causes a substant ia l delay in glucose uptake. For th i s reason, glucose was administered in so lut ion for a l l glucose tolerance tests to fol low. Another point of concern, however, was the p o s s i b i l i t y that f i s h may regurgitate the glucose s o l u t i o n . For tes t ing purposes only, a bright red food dye was added to the so lut ion to make i t v i s i b l e in water. It was then intubated into the stomachs of several f i s h . Observation of the f i s h during recovery from anaesthesia, and for 20 minutes thereaf ter , d id not reveal any evidence of regurg i ta t ion . Figure 10 : Preliminary Comparison Trial of Glucose Administration: Capsules VS Solution 100--Glucose Administration: 400 + In Solution • • In Gelatin Capsules o 300 + 200-— o Means ± standard error of the mean, N=3. + + + + + 0 ( before ) 2 3 4 5 6 7 Hours After Glucose Administration 8 cn 96 5.1.1.4 Glucose Tolerance T r i a l s This ser ies of glucose tolerance tests was conducted between November 17, 1988 and December 8, 1988. Harr i son , Robertson Creek, N i t i n a t and Big Qualicum chinook stocks were used in th i s part of the study. Each s t r a i n group was tested in dupl icate t r i a l s held approximately 1 week apart . A l l were fed a dry commercial ra t ion u n t i l commencement of the glucose tolerance t r i a l . Water temperature ranged from 8 . 6 ° C to 1 0 ° C . Before each t r i a l , feed was withheld for 63.5 to 69 hours, to ensure that a l l f i s h were in the post-absorptive s tate . The appropriate number of f i s h was captured from the holding tank and d i s t r i b u t e d among four 30 1 buckets of aerated seawater. As some stocks contained less than the idea l number of f i s h , adjustments had to be made. In some t r i a l s , c er ta in test points were excluded, or fewer f i s h were used for less c r i t i c a l sampling times. The maximum number used in any t r i a l was 36 f i s h for one r e p l i c a t e , 4 f i s h per sampling time. At the s tart of sampling 4 f i s h were anesthetized s ing ly in 2-phenoxyethanol (0.4 ml/1) and weighed. Each was k i l l e d by a sharp blow to the head and a 3 to 4 ml blood sample was taken immediately. The plasma was then separated, frozen and stored for la ter a n a l y s i s . The next 4 f i sh were s ingly anesthetized and weighed, then administered an o r a l dose of glucose (167 mg/100 g body weight). The glucose was de l ivered in so lut ion v i a syringe as described in section 5.2.2, treatment (b). The body 97 weight, glucose dose and time of intubation were recorded for each f i s h . After recovery, the 4 f i s h were placed in the f i r s t tank of the s er i e s . The procedure was continued with the remaining f i s h . The sham group was handled in the same manner as the others, except intubation was mimicked using a piece of empty tubing. The ent ire procedure took approximately 1 hours. A second t r i a l was then i n i t i a t e d , using a d i f f eren t chinook s t r a i n , with sampling times staggered accordingly . Repl icate t r i a l s were c a r r i e d out on separate days, 1 week apart . 5 .1.1.5 Experimental Sampling F i s h were captured from the f i r s t tank 3 hours af ter glucose admin i s tra t ion . They were s ingly anesthet ized, weighed and k i l l e d by a sharp blow to the head. Blood was drawn from each f i sh and handled as described in sect ion 5 .1 .1 .6 . Subsequent samplings were c a r r i e d out on the remaining groups at 6, 12, 18, 24, 30 and 36 hours re spec t ive ly , and the sham group was sampled at 12.5 hours. After every sampling, a cursory general health check was c a r r i e d out on each f i s h . A l l major organs, the kidney in p a r t i c u l a r , were inspected for les ions or other signs of in fec t ion such as b a c t e r i a l kidney disease (BKD). 5.1.1.6 Blood Sampling Technique A l l blood samples were taken from the caudal a r t e r y / v e i n just poster ior to the anal f i n . Several mis of 98 blood were c o l l e c t e d from each f i s h using a 22 gauge 1" Vacutainer needle with a 4 ml Vacutainer c o l l e c t i o n tube containing sodium heparin ant icoagulant . Samples were kept on ice no longer than 20 minutes, then centri fuged at 3,000 rpm for 10 minutes to i so la te the plasma. Each plasma sample was pipetted with a s t e r i l e disposable p ipet te into 4 p l a s t i c microcentrifuge tubes; 2 for glucose and 2 for i n s u l i n determinations. The plasma samples were immediately frozen on dry ice and stored at - 4 0 ° C for la ter a n a l y s i s . 5 .1.1.7 Plasma Glucose Determination Plasma glucose was determined using a Glucose (Trinder) Reagent K i t (Sigma K i t No. 315, Sigma Diagnost ics , St . Lou i s , MO, USA). Frozen f i s h plasma was thawed at ambient temperature. Five ul of plasma were added to 1.0 ml Glucose Trinder reagent and incubated for 18 min at room temperature. The enzymatic reac t ion , which produced a pink co lor with in tens i ty proport ional to glucose concentrat ion, proceeded as fol lows: Glucose + H 20 + 0 2 -glucose oxidase-> Gluconic ac id + H 2 0 2 H 2 0 2 + 4-Aminoantipyrine + p-Hydroxybenzene Sulphonate - p e r o x i d a s e s Quinoneimine Dye + H 20 The absorbance of the so lut ion was read in a Shimadzu UV-160 Spectrophotometer at 505 nm. Glucose concentration was ca l cu la ted from a standard curve prepared using m u l t i - l e v e l s 99 of glucose standards (Glucose/Urea Nitrogen combined standards set Cat . No. 16-11, Sigma Diagnost ics , St . Lou i s , MO, USA). 5.1.1.8 Plasma Insul in Radioimmunoassay (Conducted by Dr . E . P l i se t skaya , Dept. of Zoology, Univers i ty of Washington, Seat t l e , WA, USA) This method was a modif icat ion of Furu ich i et al. (1980), Thorpe and Ince (1976) and T h o r e l l and Larsen (1978). The f i r s t stage of th i s procedure involved preparation of unlabel led and r a d i o l a b e l l e d i n s u l i n , and a n t i - i n s u l i n serum. Salmon i n s u l i n was extracted with a c i d -acetone from fresh Langerhans i s l e t s and p u r i f i e d through a Sephadex G-50 column. Insul in standards were made by d i s so lv ing i n s u l i n in 0.1 N HCl and d i l u t e d with 0.04 M phosphate buffer to obtain concentrations of 0 to 125 MU/ml. These were used to produce standard curves (Furuichi et al., 1980). Radio label led salmon i n s u l i n was prepared by iod inatat ion with 1 2 5 i by the chloramine-T method (Hunter and Greenwood, 1962). Anti-salmon i n s u l i n serum was obtained by i n j e c t i n g rabbits with 200 jugrams of salmon i n s u l i n in Freunds complete adjuvant followed by a booster shot of 100 Mg of i n s u l i n in Freunds incomplete adjuvant after a four week i n t e r v a l . Blood was sampled from the rabbits ten days af ter the l a s t i n j e c t i o n and centrifuged at 3,000 rpm for 15 mins. Standard curves were produced by the fol lowing method: 0.1 ml of veronal buffer , salmon i n s u l i n standards (0 - 125 100 MU / ml) , ' " i - s a l m o n i n s u l i n and anti-salmon i n s u l i n serum were mixed throughly, incubated for 72 hours at 4°C and t o t a l r a d i o a c t i v i t y (t) was counted. A t o t a l of 2.5 ml of 80% ethanol was then added to each standard mixture followed by centr i fugat ion at 3,000 rpm for 15 minutes at 4°C (ethanol causes the antigen-antibody prote in complex to p r e c i p i t a t e ) . The supernatant was discarded and r a d i o a c t i v i t y of the p r e c i p i t a t e (b) counted. High b:t ra t io s were obtained from standards with low concentrations of unlabel led i n s u l i n , because l i t t l e tracer i n s u l i n was i n h i b i t e d from binding with the anti-salmon i n s u l i n serum, which p r e c i p i t a t e d out of the mixture during cen tr i fuga t ion . Standard curves were drawn from the gamma counts obtained from each i n s u l i n concentrat ion. In order to assay plasma samples, the above procedure was repeated, except that 0.1 ml of f i sh plasma was added in place of the i n s u l i n standard. Insu l in concentration was extrapolated from the standard curves (Furuichi et al., 1980). 5 .1.1.9 S t a t i s t i c a l Analyses Analyses were c a r r i e d out using the SAS analys i s of variance (ANOVA) procedure (SAS version 6.03, 1988). Part ( i ) was a nested f a c t o r i a l experiment with repeated measures. The experimental factors were s t r a i n and hour (3 chinook s tra ins x 9 sampling hours) and experimental rep l i ca te s were nested within each s t r a i n and hour. The 101 i n d i v i d u a l measurements taken within an experimental unit (tank) represented repeated measurements. (Snedecor and Cochran, 1980) Data from the Big Qualicum chinook were not included in the analys i s due to presence of B a c t e r i a l Kidney Disease in the stock. 5.2 Part i i ) Oral Glucose Tolerance in Chinook Salmon Acclimated to High and Low Carbohydrate Diets Respect ive ly . 5.2.1 MATERIALS AND METHODS 5.2.1.1 Glucose Tolerance T r i a l s This ser ies of glucose tolerance tests was conducted using Capilano and Quinsam chinook stocks. T r i a l s were c a r r i e d out between December 8 and 19, 1988, and water temperature ranged from 8 . 2 ° C to 9 ° C . The Capilano and Quinsam stocks were each d iv ided into 2 groups (in two 8' diameter tanks) and acclimated to the 2 test d ie ts used in experiment 1. One group was fed the contro l (low carbohydrate) d iet and the other was fed the high carbohydrate diet for two weeks p r i o r to t e s t i n g . Duplicate glucose tolerance t r i a l s were conducted on each of the diet groups of Capilano chinook, approximately one week apart . The experimental protocol and assay techniques used in th i s study were s imi lar to those used in part ( i ) (refer to sections 5.2.3 through 5 .2 .7 ) . 102 Quinsam chinook had to be el iminated from the experiment due to the presence of B a c t e r i a l Kidney Disease in the stock, which became evident during tolerance t e s t ing . 5.2.1.2 S t a t i s t i c a l Analyses Analyses were c a r r i e d out using the SAS analys i s of variance (ANOVA) procedure (SAS version 6.03, 1988). Part ( i i ) was a nested f a c t o r i a l experiment with repeated measures. The experimental factors were diet and hour (2 accl imation d ie t s x 9 sampling hours) and experimental r ep l i ca te s were nested within each s t r a i n and hour. The i n d i v i d u a l measurements taken within an experimental unit (tank) represented repeated measurements. 103 5.3 RESULTS 5.3.1 Part i ) Oral Glucose Tolerance in Chinook Salmon of Selected Strains S i g n i f i c a n t d i f ferences in response to the ora l glucose tolerance test between s t ra ins could be discussed only in terms of the o v e r a l l response curves. Indiv idual test points ( s tra in/hour means) could not be d is t inguished s t a t i s t i c a l l y due to high v a r i a t i o n and small sample s i ze s . Plasma glucose and i n s u l i n l eve l s of the 12.5 hour sham groups were not s i g n i f i c a n t l y d i f f erent from res t ing l eve l s (pr ior to t e s t i n g ) . Table 16 and Figure 11 show plasma glucose and i n s u l i n l eve l s in chinook salmon of three s t r a i n s , before and af ter adminis trat ion of the o r a l glucose tolerance test (GTT). Average body weight (± 1 s .d . ) of the f i s h tested were 188 ± 73 g in Harrison chinook, 215 ± 51.4 g in Robertson Creek chinook, and 263 ± 8 7 g in N i t i n a t chinook. Body weight was confounded with f i sh s t r a i n , and i t s e f fect could not be adjusted for in the analyses (see d i scuss ion) . 5.3.1.1 Plasma Glucose Response In the analys is of plasma glucose response to o r a l glucose chal lenge, there was a s i g n i f i c a n t in terac t ion between s t r a i n and hour (p<0.05) in plasma glucose l e v e l s , i n d i c a t i n g a di f ference in the response pattern over time between chinook s t r a i n s . 104 Table 16 - Mean plasma glucose and i n s u l i n concentrations in 3 s trains of chinook salmon subjected to an o r a l glucose tolerance test (GTT). S tra in : Harrison Robertson C r . N i t i n a t Hour (post GTT) 0 G l u e 2 76 + 5.4 70 + 1 .9 80 + 4.6<6) (before) Ins 3 2. 8 ± . 2 6 4. 1 ± . 1 4 Sham Glue 78 + 7.5 70 + 3.0 72 + 1.8<6> (12.5) Ins 2. 9 ± . 2 8 4. 2 ± . 3 2 3 Glue 155 + 22.7<4> 157 + 10.6 177 + 5.8<4> Ins 2. 4 ± . 1 2 4. 6 ± . 6 3 6 Glue 252 + 21 .8 244 + 9.8 239 + 11.0 Ins 4. 4 ± . 5 0 5. i ± . 6 4 12 Glue 308 + 20.0 366 + 25.7 385 + 15.0 Ins 4. 2 ± . 6 4 7. 1 ± . 4 0 18 Glue 338 + 37.5 436 + 22.4 495 + 25.0 Ins 4. 6 ± . 6 2 6. 6 ± . 5 6 24 Glue 269 + 17.3 360 + 23.5 402 + 38.7 Ins 4. 0 ± . 2 7 5. 9 ± . 6 2 30 Glue 190 + 24.0 328 + 34.8 340 + 57.7 Ins 3. 6 ± . 6 0 5. 9 ± . 5 3 36 Glue 188 + 25.9 215 + 18.2 263 + 30.9 Ins 4. 1 ± . 6 4 5. 9 ± . 4 9 Avg. Weight (g ) 4 180. 8 ± 2 9 . 5 295. 3 ±61 .6 389 .8 ± 5 0 . 1 1 Plasma i n s u l i n was not assayed in the N i t i n a t s t r a i n . 2 Mean plasma glucose concentration ± SEM (standard error of the mean). There was a s i g n i f i c a n t in terac t ion between diet and s tra in in plasma glucose response (p<0.05) (see text for explanation) . 3 Mean plasma i n s u l i n concentration ± SEM. Plasma i n s u l i n response was s i g n i f i c a n t l y d i f f erent between Harrison and Robertson Creek chinook (p<0.000l) (see text for explanat ion) . Each plasma glucose and i n s u l i n value was obtained using 8 f i s h except where indicated in brackets , beside glucose values . 4 Average body weight of a l l f i sh used in t r i a l ± 1 standard d e v i a t i o n . 105 Figure 11: Oral Glucose Tolerance Test Response in Chinook Salmon of Selected B. C. Strains. 600 means ± 1 s.e.rrJ H 1 1— 0 3 6 HARRISON H 1 1 h H H + 12 18 24 • • ROB. CRK. 30 36 — - NITINAT c z to < 2 HOURS AFTER GLUCOSE ADMINISTRATION 1 Means ± 1 standard error of the mean, N=8. 106 Mean plasma glucose concentrations ranged from 70 - 80 mg/dl in chinook sampled just p r i o r to the challenge and in sham groups sampled at 12.5 hours af ter mock t e s t i n g . Plasma glucose concentrations peaked at 18 hours post challenge in each of the three chinook s tra ins tested. The peak mean of plasma glucose at the 18th hour was 338 mg/dl in Harrison chinook, 436 mg/dl in Robertson Creek chinook, and 495 mg/dl in N i t i n a t chinook. In r e l a t i v e terms, the glucose peak in Harrison chinook was 77% that of Robertson Creek chinook and 68% that of N i t i n a t chinook. At 36 hours post glucose chal lenge, mean plasma glucose concentrations in the Harr i son , Robertson Creek and N i t i n a t chinook remained subs tant ia l ly e levated, at 188 mg/dl , 215 mg/dl and 263 mg/dl re spec t ive ly . Body weight and plasma glucose concentrat ion, determined within each sampling time (hour), were poorly corre la ted within s t r a i n s . 5.3.1.2 Plasma Insu l in Response Plasma i n s u l i n response to the ora l glucose challenge was s i g n i f i c a n t over time (p<0.05), and s i g n i f i c a n t l y higher o v e r a l l in Robertson Creek chinook than in Harrison chinook (p<0.000l). Plasma i n s u l i n peaked at the 12th hour in Robertson Creek chinook (7.1 ng/ml) and the 18th hour in Harrison chinook (4.6 ng/ml) , although these peaks were very i n d i s t i n c t (see Figure 10). Resting plasma i n s u l i n concentrations (in p r e - t r i a l and sham groups) were also 107 higher in Robertson Creek chinook, averaging 4.15 ng/ml, as compared to 2.85 ng/ml in Harrison chinook. Insul in data for the N i t i n a t s t r a i n were not a v a i l a b l e . Body weight and plasma i n s u l i n concentrat ion, computed within each sampling time, were poorly corre la ted within s t r a i n s . Poor c o r r e l a t i o n was also found between plasma glucose and i n s u l i n concentrat ion, within each sampling time. 5.3.2 Part i i ) Ef fect of Pre- test Diet on Oral Glucose Tolerance in Capilano Chinook Salmon Although th i s study was conducted on both Capilano and Quinsam chinook stocks, the l a t t e r had to be e l iminated from analyses due to a high incidence of B a c t e r i a l Kidney Disease. S i g n i f i c a n t dif ferences in response to the o r a l glucose tolerance test between diet groups could be discussed only in terms of the o v e r a l l response curves. Indiv idual test points (diet /hour means) could not be d is t inguished s t a t i s t i c a l l y due to high v a r i a t i o n and small sample s i ze s . Table 17 and Figure 12 show plasma glucose and i n s u l i n concentrations in Capilano chinook pre-accl imated to e i ther low (control) or high carbohydrate d i e t , before and after administrat ion of the o r a l glucose tolerance test (GTT). 108 Table 17 - Mean plasma glucose and i n s u l i n concentrations in Capilano chinook salmon subjected to an o r a l glucose tolerance test (GTT) af ter feeding low or high carbohydrate pre- tes t d ie t s re spec t ive ly . Diet : Low C a r b . 1 High C a r b . 1 Hour (post GTT) 0 G l u e 2 65 ± 1.7(5) 68 ± 6 . 0 ( 5 ) (before) Ins 3 5.6 + 4.3 ± . 7 0 Sham Glue 61 ± 6.3(3) 61 ± 4 .0 ( 3) (12.5) Ins 3.9 ± . 4 7 5.0 ± . 2 7 3 Glue 175 ± 18.6(3) 165 ± 15.8 ( 3) Ins 3.9 ±1 .05 4.5 ± . 9 1 6 Glue 267 ± 5.6 217 ± 31.9 Ins 10.5 ±1 .40 5.8 ± 1 . 3 9 12 Glue 388 ± 16.9 302 ± 24.6 Ins 9.1 ± 1 . 1 0 8.8 ± . 8 0 18 Glue 464 ± 34.0 336 ± 30.8 Ins 6.6 ± . 2 5 9.2 ± . 5 1 24 Glue 457 ± 3 7 . 9 ( 4 ) 342 ± 3 3 . 1 ( 4 ) Ins 7.4 ± . 3 8 7.6 ± 1 . 1 8 30 Glue 358 ± 34.7 206 ± 30.8 Ins 5.3 ± . 4 8 5.7 ± . 7 7 36 Glue 250 ± 7.1<4) 211 ± 3 8 . 7 ( 4 ) Ins 4.6 ± . 5 6 5.0 ± 1 . 0 1 Average weight (g ) 4 399.7 ± 75.9 390.0 ± 69.9 Low. (control) and high carbohydrate d ie t s from experiment 1 (see table 1 for compositional information) . Mean plasma glucose concentration ± SEM (standard error of the mean). O v e r a l l plasma glucose response was s i g n i f i c a n t l y d i f f erent between diet groups (p<0.00l). Mean Plasma i n s u l i n concentration ± SEM. Overa l l plasma i n s u l i n response was not s i g n i f i c a n t l y d i f f erent between diet groups (see text for explanat ion) . Each plasma glucose and i n s u l i n value was obtained using 7 f i s h except where indicated in brackets beside glucose values . Average body weight of a l l f i sh used in t r i a l ± 1 standard dev ia t ion . 109 Figure 12: Effect of Pre-test Diet on Oral Glucose Tolerance Test Response in Capilano Chinook Salmon 600 E LU CO o o 20 c 3 CO z 15 + 10 + 5 + 0 3 6 12 — LOW CARB. Diet 24 30 36 • •• HIGH CARB. Diet means ± 1 s.e.m. H 1 1 1 h H h—I h 0 3 6 before 12 18 24 30 36 HOURS AFTER GLUCOSE ADMINISTRATION 1 Means ± 1 standard error of the mean, N=7. 110 5.3.2.1 Plasma Glucose Response Plasma glucose concentrations were s i g n i f i c a n t l y higher (p<0.001) in f i s h fed the low carbohydrate (control) d ie t than in those fed the high carbohydrate d iet p r i o r to t e s t i n g . Plasma glucose peaked at 18 hours post glucose challenge in both diet groups, with peak means reaching 464 mg/dl in contro l -acc l imated f i s h and 336 mg/dl in high carbohydrate-acclimated f i s h . In r e l a t i v e terms, the peak in plasma glucose concentration of chinook acclimated to the high carbohydrate diet was 72.5% that of contro l d i e t -acclimated f i s h . Plasma glucose concentrations were cons i s tent ly lower in the high carbohydrate d iet group (throughout the t r i a l ) . Plasma glucose was poorly corre la ted with body weight within the i n d i v i d u a l sampling times in each diet group. 5.3.2.2 Plasma Insul in Response Plasma i n s u l i n concentrations changed s i g n i f i c a n t l y over time (p<0.05), but d id not d i f f e r s i g n i f i c a n t l y between the two diet groups. Although there was no s i g n i f i c a n t in terac t ion between diet and hour in plasma i n s u l i n concentrat ion, the i n s u l i n response patterns appear to d i f f e r between diet groups. Insu l in concentrations peaked in the contro l group at the 6th hour, at a mean concentration of 10.5 ng/ml, and in the high carbohydrate group at the 18th hour, at a mean l e v e l of 9.2 ng/ml. Insul in concentrations were s imi lar in the two diet groups I l l at 12 hours post chal lenge, averaging 9.1 ng/ml and 8.8 ng/ml in c o n t r o l - and high carbohydrate-acclimated groups re spec t ive ly . Nevertheless these 'apparent' di f ferences were not s i g n i f i c a n t , poss ib ly due to high v a r i a t i o n in the data and small sample s i ze s . Poor corre la t i ons were found between plasma i n s u l i n and body weight, within each diet group, at a given sampling hour. Plasma glucose and i n s u l i n concentrations were also poorly c o r r e l a t e d . 112 5.4 DISCUSSION 5.4.1 O r a l Glucose T o l e r a n c e : General O r a l glucose t o l e r a n c e t e s t i n g was chosen over other approaches because i t i n v o l v e s a normal p h y s i o l o g i c a l route of glucose a d m i n i s t r a t i o n . In humans, through i t s e f f e c t s on g a s t r o i n t e s t i n a l hormones, the o r a l d e l i v e r y of glucose evokes a g r e a t e r i n s u l i n response than intravenous a d m i n i s t r a t i o n ( C a h i l l , 1971). B e t t e r c o r r e l a t i o n has been shown between the o r a l GTT and other i n d i c e s of glucose d i s p o s a l , than with the IV t e s t (Wolfe et al., 1978). In t h i s s e r i e s of glucose t o l e r a n c e t e s t s , a c a l c u l a t e d dose of 167 mg glucose / 100 g body weight was d e l i v e r e d o r a l l y i n an 80% glucose s o l u t i o n by way of stomach i n t u b a t i o n . The dosage used i n these t r i a l s was the same as that used by Wilson and Poe (1987) in channel c a t f i s h , and by F u r u i c h i and Yone i n carp, red sea bream and y e l l o w t a i l (1981, 1982a, 1982c). I t i s e q u i v a l e n t to the 100 g / 60 kg body weight dose commonly used i n human t r i a l s . In the s t u d i e s mentioned p r e v i o u s l y , glucose was d e l i v e r e d to the f i s h o r a l l y i n g e l a t i n c a p s u l e s . In t h i s study, i t was r e v e a l e d i n the p r e l i m i n a r y t r i a l that f i s h given glucose v i a c a p s u l e s e x h i b i t e d a lower e l e v a t i o n of plasma glucose i n comparison to f i s h given the sugar i n s o l u t i o n . The time r e q u i r e d f o r enzymatic breakdown of the capsule w a l l , delayed the uptake of glucose i n t o the c i r c u l a t i o n . Capsule d i g e s t i o n t h e r e f o r e i n t r o d u c e d a 113 confounding factor in the tolerance t e s t i n g . The fact that glucose powder had to be administered in more than one capsule, due to i t s bulk, accentuated the problem. In a d d i t i o n , the wider tubing required for o r a l de l ivery of capsules presented a greater r i sk of trauma to f i s h than the fine cannula tubing used for intubating s o l u t i o n . For these reasons, the glucose so lu t ion- in tubat ion method was chosen. A major concern, however, in using the so lu t ion- in tubat ion method was the p o s s i b i l i t y of r egurg i ta t ion , as f i s h recovering from anaesthesia displayed a 'cough- l ike ' response. Intubation of glucose so lut ion containing a bright food dye for v i s i b i l i t y in water d id not reveal regurgi ta t ion within the f i r s t 20 minutes post recovery. During these tolerance t r i a l s seawater temperature ranged from 8.2 °C to 10 °C. Repl icate t r i a l s were c a r r i e d out on separate days, approximately one week apart , in order to e l iminate b ia s . Temperature must be taken into account when assessing glucose tolerance response. As metabolic rate decl ines with decreasing temperature, poorer glucose tolerance would be expected at lower temperatures. Handling stress has been shown to cause hyperglycaemia in chinook salmon, which pers i s t s for many hours (Barton and Schreck, 1988; Soiv io and O i k a r i , 1976). Sham groups were included in the GTT in order to assess the effect of the procedure alone on blood glucose. Sham groups sampled at 2, 6 and 12 hours after mock treatment in a prel iminary tes t , and at the 12.5 hours in actual t r i a l s , d id not exhibi t 114 elevat ions in plasma glucose concentration exceeding 100 mg/dl . 5.4.1.1 Part i ) Oral Glucose Tolerance in Chinook Salmon of Selected Stra ins In th i s examination of glucose tolerance in chinook of d i f f erent s t r a i n s , there are two major points that must be addressed. F i r s t , the chinook s tra ins used in these t r i a l s were d i s s i m i l a r in body weight (see Table 16). Metabolism is known to be weight dependent, and thus can be expected to exert some effect on glucose to lerance . As body weight i s associated with chinook s t r a i n , i t i s a confounding f a c t o r . Due to the var iab le nature of the data, and poor corre la t i ons between body weight and plasma glucose l e v e l within i n d i v i d u a l s t r a i n s , covariance analys i s could not be app l i ed . In order to minimize the ef fects of s i ze , glucose was administered on a body weight bas i s , therefore a l l chinook received the same r e l a t i v e dosage. Nevertheless, because of the di f ferences in mean body weight of the s t r a i n s , conclusions regarding genetic di f ferences in glucose tolerance are not warranted. Second, the ascending, port ion of the glucose tolerance response curve expresses the balance between the rate of glucose uptake and i t s subsequent d isposal at each sampling time. It i s d i f f i c u l t to e s tab l i sh glucose absorption rates from such response curves. 115 The plasma glucose concentrations in these chinook t r i a l s are comparable to those found in rainbow trout (Palmer and Ryman, 1972) and are ind ica t ive of poor glucose to lerance . Plasma glucose curves were s imi lar up to the 6th hour af ter the glucose chal lenge, at which time they began to diverge. Plasma glucose peaked at 18 hours post glucose challenge in each of the Harr i son , Robertson Creek and N i t i n a t chinook s t r a i n s , reaching l eve l s of 338 mg/dl , 436 mg/dl and 495 mg/dl , re spec t ive ly , and remained elevated at 36 hours after the challenge (see Figure 11). Continued plasma glucose e levat ion in a l l s t ra ins at 36 hours after glucose administrat ion poss ibly re f l ec ted some degree of hyperglycaemia associated with s tress and with the fact that feed had been withdrawn for a period of approximately 100 hours at th i s time. The glucose challenge e l i c i t e d a gradual i n s u l i n response with i n d i s t i n c t peaks of 4.63 ng/ml and 7.07 ng/ml in Harrison and Robertson Creek chinook, re spec t ive ly . The r i s e in plasma i n s u l i n appeared to commence at the s ix th hour, continue through the 12th and 18th hours, then decl ined gradual ly . At the 36th hour, plasma i n s u l i n concentrations also remained e levated. Resting plasma i n s u l i n l eve l s were in the range of 2 to 5 ng/ml, s imi lar to those found in other f i s h species , but high in comparison to res t ing l eve l s in humans. The r e l a t i v e increase in plasma i n s u l i n over fas t ing concentration was less than 2 - f o l d . By contrast , humans 116 exhibi t 8 to 10-fold increases in plasma i n s u l i n within 30 to 60 minutes after a glucose challenge ( C a h i l l , 1971). The magnitude and timing of the i n s u l i n peaks, and poor corre la t ions between plasma i n s u l i n and plasma glucose are consistent with reports by other invest igators regarding glucose as a stimulant for i n s u l i n release in salmonids. As has been'discussed in the L i t e r a t u r e Review, the primary stimulus for i n s u l i n secret ion in f i sh i s plasma amino ac ids . It i s in teres t ing that the plasma glucose response of the smaller Harrison chinook was lower than that of the larger chinook of Robertson Creek and N i t i n a t s t r a i n s , e spec ia l ly in view of the fact that carbohydrate metabolism is reported to increase with f i s h s i z e . This may be ind ica t ive of a s t r a i n di f ference in glucose u t i l i z a t i o n . However, di f ferences in absorptive capac i t i e s cannot be ruled out. If plasma glucose.concentrations are indeed lower in Harrison chinook, i t cannot be a t t r ibuted to an enhanced i n s u l i n response. 117 5.4.2.1 Part i i ) Ef fec t of Pre-Test Diet on Oral Glucose Tolerance The accl imation d ie t was demonstrated to have a s i g n i f i c a n t effect on plasma glucose response. Chinook fed the high carbohydrate d iet for two weeks p r i o r to adminis trat ion of a glucose dose exhibited 27.5 % lower peak plasma glucose concentrations than were evident in chinook fed the contro l d i e t . Plasma glucose peaked 342 mg/dl in high carbohydrate-fed chinook and at 434 mg/dl in c o n t r o l -fed chinook, with highest l eve l s observed at 1 8 - 2 4 hours post-glucose chal lenge. This is ind ica t ive of an adaptation response to the high carbohydrate d i e t , although hyperglycaemia was s t i l l pronounced and p e r s i s t e n t . There appeared to be a di f ference in the i n s u l i n response patterns in the two diet groups, although the d i f ference i s d i f f i c u l t to evaluate. In comparison to the i n s u l i n responses observed in part i ) , i n s u l i n concentrations appear higher in th i s study. The Capilano chinook in th i s t r i a l had a higher average body weight 390 g) and higher res t ing plasma i n s u l i n l eve l s 5 ng/ml) . Their i n s u l i n response to glucose challenge represented a 2-fo ld increase in concentrat ion. Based on his 1987 study, H i l t o n postulated that rainbow trout are s imi lar to type - I I , rather than type-I , d i a b e t i c s . In other words, the ir d i a b e t i c - l i k e nature i s not due to a def ic iency of i n s u l i n , but rather , some other i n h i b i t i n g factor (Hi l ton reported an increase in plasma i n s u l i n in 118 f i s h fed a high carbohydrate diet and concluded therefore that elevated plasma glucose was not due to i n s u f f i c i e n t i n s u l i n ) . 119 5.5 CONCLUSIONS (Experiment 2) Pronounced and pers i s tent hyperglycaemia in response to o r a l glucose tolerance tes t ing revealed poor glucose tolerance in chinook salmon of the s t ra ins examined. Plasma glucose concentrations fol lowing glucose administrat ion increased 5 to 9 times over i n i t i a l concentrat ions . The three chinook s tra ins compared showed di f ferences in glucose tolerance and there was also a s t r a i n di f ference in c i r c u l a t i n g i n s u l i n concentrat ion. No r e l a t i o n s h i p was however evident between glucose tolerance and plasma i n s u l i n concentrat ion. Accl imation to a high carbohydrate d iet induced some improvement in glucose to lerance , as indicated by a 30% decl ine in peak plasma glucose concentrat ions . Poor c o r r e l a t i o n between plasma glucose and i n s u l i n concentrations indicates that glucose is a poor i n s u l i n secretagogue. The improvement in glucose tolerance demonstrated in th i s experiment supports the evidence of adaptation obtained in Experiment 1, in which f i s h i n i t i a l l y responded to imposition of a high carbohydrate d iet by a reduction in growth rate , but subsequently adapted and grew at a rate comparable to that of f i s h fed the contro l d i e t . Although i t appears that there may be some genetic di f ferences in glucose to lerance , the evidence should not be regarded as conc lus ive . Oral glucose tolerance may serve as a useful "screening-test" in examining glucose u t i l i z a t i o n . 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